WO2014151030A1

WO2014151030A1 - Cell proliferation inhibitors and conjugates thereof

Info

Publication number: WO2014151030A1
Application number: PCT/US2014/024795
Authority: WO
Inventors: Paul A. Barsanti; Sylvie Chamoin; Lionel DOUMAMPOUOM-METOUL; Bernhard Hubert GEIERSTANGER; Robert Martin GROTZFELD; Stephanie GUERRO-LAGASSE; Darryl Brynley JONES; Alexei Karpov; Marc LAFRANCE; Cristina NIETO-OBERHUBER; Weijia Ou; Grazia Piizzi
Original assignee: Novartis Ag; Irm Llc
Priority date: 2013-03-15
Filing date: 2014-03-12
Publication date: 2014-09-25
Also published as: JP2016516035A; EP2968591A1; CN105451773A; TW201512174A; UY35471A

Abstract

Disclosed herein are immunoconjugates comprising an inhibitor of Eg5 linked to an antigen binding moiety such as an antibody, useful for treating cell proliferative disorders. Also disclosed are novel inhibitors of Eg5 that can be used either alone or as part of an immunoconjugate to treat cell proliferation disorders. The Eg5 inhibitors include compounds of this formula as described herein: [insert last structure from page 68 here] The invention further provides pharmaceutical compositions comprising these compounds and immunoconjugates, optionally including a therapeutic co-agent, and methods to use these compounds, conjugates and compositions for treating cell proliferation disorders.

Description

CELL PROLIFERATION INHIBITORS AND CONJUGATES THEREOF

FIELD OF THE INVENTION

The invention provides compounds that inhibit cell proliferation by inhibiting Eg5 activity, and are thus useful to treat cellular proliferative disorders associated with excessive Eg5 activity. The invention also includes conjugates that comprise an inhibitor of Eg5 linked to an antigen-binding moiety, and pharmaceutical compositions containing these conjugates. Also included are methods of using these compounds and conjugates to treat cell proliferation disorders, including cancers.

BACKGROUND

In recent years, a great deal of research has been directed toward the use of antibodies to deliver cell proliferation inhibitors and cytotoxic agents to specific cells that are targeted for elimination, by forming antibody-drug conjugates (ADCs). The ADCs typically contain an antibody selected for its ability to bind to a cell targeted for therapeutic intervention, linked to a drug selected for its cytostatic or cytotoxic activity. Binding of the antibody to the targeted cell delivers the drug to the site where its therapeutic effect is needed and thus reduces off-target activities while improving the efficiency with which the payload compound is utilized.

Many antibodies that recognize and selectively bind to targeted cells, like cancer cells, have been disclosed for use in ADCs, and many methods for attaching payload (drug) compounds such as cytotoxins to antibodies have also been described. In spite of the extensive work on ADCs, though, only a few classes of cell proliferation inhibitors or cytotoxins have been used extensively as ADC payloads. Even though the first ADC approved for use in humans in the U.S. was launched in 2000 (Mylotarg®, which was later withdrawn from the market), a decade later only a few chemical classes of drug compounds (maytansinoids, auristatins, calicheamycins and duocarmycins) had reached clinical trials as payloads for ADCs. Antibody-Drug Conjugates: the Next Generation of Moving Parts, A. Lash, Start-Up, Dec. 201 1 , 1-6. This suggests how difficult it is to identify a suitable class of drug compounds that make effective ADC payloads. Given the widely acknowledged value of ADCs as therapeutics, particularly for treating cancer, there thus remains a need for novel cell proliferation inhibitors suited for use as payloads in ADCs. SUMMARY OF THE INVENTION

The invention includes novel inhibitors of Eg5, and methods of using Eg5 inhibitors either as small-molecule pharmaceuticals or as the drug component (payload) of an antibody-drug conjugate (ADC).

Eg5, also known as kinesin spindle protein or KSP, is a kinesin motor protein, involved in cross-linking of microtubules during mitosis, and is thus required for cell division. Inhibitors of Eg5 are known to be useful to treat cell proliferation disorders like cancer (Rath and Kozielski, Nature Rev. Cancer, vol. 12, 527-39 (2012); see also WO06/002236, WO2007/021794, WO2008/063912, WO2009/077448, WO201 1/128381 , WO201 1/128388, and WO2006/049835). While a number of different chemical families of Eg5 inhibitors are known, they have not heretofore been used in ADCs. The present invention includes use of Eg5 inhibitors as drug payloads for ADCs, and novel Eg5 inhibitors that are useful as ADC payloads and as small-molecule pharmaceuticals. The invention further includes methods and intermediates useful for incorporating certain Eg5 inhibitors into ADCs, and methods to use the novel compounds and conjugates to treat cell proliferation disorders.

The present invention provides immunoconjugates (e.g., ADCs) containing inhibitors of Eg5 linked to an antigen binding moiety such as an antibody or antibody fragment. These conjugates comprising an Eg5 inhibitor are useful to treat cell proliferation disorders, particularly when the Eg5 inhibitor is linked to an antibody that recognizes cancer cells and thus promotes delivery of the Eg5 inhibitor to a cancer cell targeted for attack. The immunoconjugates are especially useful for treating certain cancers as further detailed herein. Data provided herein demonstrate that these immunoconjugates are effective inhibitors of cell proliferation and for treating some types of cancer; without being bound by theory, it is believed their activity is due to inhibition of Eg5 in cells.

In one aspect, immunoconjugates of the invention include compounds of this formula:

wherein Ab represents an antigen binding moiety;

L represents a linking group that connects X to Ab;

m is an integer from 1 -4;

n is an integer from 1 to 16; and

X independently at each occurrence represents an inhibitor of Eg5. Where m is greater than 1 , each L is independently selected. In some embodiments, each L is the same.

In these immunoconjugates, X can be a compound of Formula II as described herein, or any Eg5 inhibitor having an IC-50 below about 100 nM for inhibition of Eg5. Many such Eg5 inhibitors are known, including ispinesib, SB-743921 , AZD4877, ARQ621 , ARRY-520, LY2523355, MK-0731 , EMD534085, and GSK-923295, and Eg5 inhibitors described in WO06/002236, WO2007/021794, WO2008/063912, WO2009/077448, WO201 1/128381 , WO201 1/128388, and WO2006/049835.

Typically, m is 1 or 2 in immunoconjugates of this formula, preferably 1 ; and n is 2-8, preferably about 2 to about 6, more preferable between 3 and 5. Ab can be any suitable antigen binding moiety, and is often an antibody. Suitable antibodies are well known in the art, and may be either native antibody sequence or they may be modified by, e.g., protein engineering techniques to improve their usefulness or activity. L can be any linker suitable for attaching one or more X groups to Ab; often L is attached to a lysine delta- amino group, or to a cysteine sulfhydryl of the antibody. These can be naturally-occurring residues, or they can be introduced at selected locations in the antibody sequence.

Suitable options for X include compounds of Formula (II) disclosed herein, as well as monastrol (Ethyl 4-(3-hydroxyphenyl)-6-methyl-2-sulfanylidene-3,4-dihydro-1 H- pyrimidine-5-carboxylate); (2S)-4-(2,5-Difluorophenyl)-N-[(3R,4S)-3-fluoro-1 -methyl-4- piperidinyl]-2,5-dihydro-2-(hydroxymethyl)-/V-methyl-2-phenyl-1 H-pyrrole-1-carboxamide (MK-0731 , CAS 845256-65-7); Litronesib (LY2523355, CAS 910634-41 -2); and (2S)-2- (3-Aminopropyl)-5-(2,5-difluorophenyl)-/V-methoxy-/V-methyl-2-phenyl-1 ,3,4-thiadiazole- 3(2H)-carboxamide (ARRY520); and AZ3146 (9-Cyclopentyl-2-[[2-methoxy-4-[(1 - methylpiperidin-4-yl)oxy]-phenyl]amino]-7-methyl-7,9-dihydro-8/-/-purin-8-one).

In certain embodiments, the immunoconjugate is of Formula (I)

(I)

wherein Ab represents an antigen binding moiety such as an antibody or antibody fragment;

L represents a linking group that connects X to Ab by covalent or non-covalent bonding, which may optionally attach more than one X to Ab, and which may or may not be designed to facilitate in vivo cleavage;

X independently at each occurrence represents a compound of Formula (II):

(II)

as further described below; m is an integer from 1-4, typically 1-2; and n is an integer from 1 to 16, preferably 2-8.

Where more than one linking group L is present, each L can be independently selected. In some embodiments, each group L is the same.

The invention provides methods for making ADCs using Eg5 inhibitors, particularly compounds of Formula (II) or (III), as the payload (drug) to be delivered, and methods to use these ADCs to treat cell proliferation disorders.

The invention also provides modified compounds of Formula (II) that are described herein as Formula (IIA) and (MB) and (IIC): these are structures that comprise a compound of Formula (II) having a reactive functional group and optionally one or more linker components attached, to facilitate connecting the compound either directly or indirectly to an antibody or antigen binding fragment. These compounds are useful to make immunoconjugates. Thus, in another aspect, the invention provides compounds of Formula (IIA) and (MB) and (110):

wherein W comprises a reactive functional group that can be used to connect (IIA) or (MB) or (IIC) to a linker component, or directly to Ab, to provide an immunoconjugate of Formula (I), and methods to use these compounds for making ADCs.

In another aspect, the invention provides novel Eg5 inhibitors of Formula (III) as described herein and pharmaceutically acceptable salts thereof.

These compounds are novel inhibitors of Eg5 and possess anticancer activity as shown herein. They can be used as ADC payloads as demonstrated herein, or, like other inhibitors of Eg5, they can be used as small-molecule therapeutic agents for treatment of cell proliferation disorders.

In another aspect, the invention provides pharmaceutical compositions comprising an immunoconjugate of Formula (I) or a compound of Formula (III) admixed with at least one pharmaceutically acceptable carrier or excipient, optionally admixed with two or more pharmaceutically acceptable carriers or excipients, and methods to use these

compositions to treat cell proliferation disorders.

In another aspect, the invention provides a method to treat a condition

characterized by excessive or undesired cell proliferation, which comprises administering to a subject in need of such treatment an effective amount of an immunoconjugate of Formula (I) or a compound of Formula (III), or any subgenus thereof as described herein, or a pharmaceutical composition comprising such compound or immunoconjugate. The subject for treatment can be a mammal, and is preferably a human. Conditions treatable by the compounds and methods described herein include various forms of cancer, such as gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, renal, gastric (e.g., gastrointestinal stromal tumors (GIST)), liver, and pancreatic cancer, and sarcoma. Other cell proliferation disorders that can be treated with these methods and compositions include diabetic retinopathy, liver and lung fibrosis, Sjogren's syndrome, and lupus erythematous.

The invention includes compositions of Formulas (l)-(lll) and the subgenera thereof as described herein, and all stereoisomers (including diastereoisomers and enantiomers), tautomers and isotopically enriched versions thereof (including deuterium substitutions) as well as pharmaceutically acceptable salts of these compounds.

Compositions of the present invention also comprise polymorphs of Formula (l)-(lll) (or sub-formulas thereof) and salts, particularly pharmaceutically acceptable salts, thereof.

In another aspect, the invention provides immunoconjugates comprising an improved linking group that connects an antibody with a payload such as a cytotoxin, including the Eg5 inhibitors described herein. These immunoconjugates comprise a linking group that comprises a group of the formula -C(0)NR - or -NR -C(O)-, which may be an amide or a carbamate, wherein R²¹ is of the formula -(CH₂)i-4-R²², R²² is a polar group selected from -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k- OCH₂CH₂OR²³, and -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or C1-4 alkyl. These linking groups reduce aggregation of the immunoconjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1 B. Determination of average drug loading (DAR, drug to antibody ratio) for an ADC based on heavy chain and light chain loading.

FIG. 2A-2E. Antiproliferative activity of various compounds of Formula (II) and (III) in cell cultures.

FIG. 3A-3L. Anti-proliferative activities of certain Eg5 inhibitors across a variety of cancer cell lines derived from different lineages.

FIG. 4A-4V. In vitro anti-proliferative activity of ADCs on a cell line engineered for high Her2 expression vs. matched parental (Her2-low) cell line.

FIG. 5A - 5E. In vitro anti-proliferative activity of ADCs on cell lines with endogenous Her2 expression.

FIG. 6(A) Efficacy of a TBS-Cmpd 220 conjugate in HCC 1954 breast cancer xenografts.

FIG. 6(B). Efficacy of a TBS-Cmpd 220 conjugate in HCC 1954 breast cancer xenografts.

FIG. 7 (A) and (B) shows efficacy of a TBS-Cmpd 220 conjugate in SK-OV-3ip xenografts.

FIG. 8 shows efficacy of a TBS-Cmpd 215 conjugate in SK-OV-3ip xenografts.

FIG. 9 shows efficacy of a TBS-Cmpd 223 conjugate in SK-OV-3ip xenografts.

FIG. 10(A) shows the degree of aggregation of construct referred to as ADC-1 10 as measured by size exclusion chromatography. The amount of aggregate detected is about 12% of the total detected conjugate.

FIG. 10(B) shows the amount of aggregation of ADC-1 1 1 , which is about 2.4%. FIG. 10(C) shows the amount of aggregation of ADC-1 12, which is about 2.7%. FIG. 11 shows in vitro activity of ADC-1 10 and ADC-1 1 1 against various cell types.

Fig. 12 shows activity of a series of immunoconjugates having different payloads (5A, 5B, 5C, 5D, 5E and 5F from Table 5) linked to antibody cKitA. All exhibit good to excellent activity in cell culture on SK-OV-3ip.

Fig. 13 shows activity of selected payloads conjugated to trastuzumab (TBS) on various tumor cell lines, demonstrating that a variety of payloads of Formula I I are active against various cancer cell lines. The payload compounds are found in Tables 5 and 6.

Fig. 14 shows a representative inhibitor of the invention compared to Eg5 inhibitors from other compound classes; compounds in Figure 14 are all linked to trastuzumab via a Val- Cit linker.

Fig. 15 shows activity of trastuzumab immunoconjugates of the invention on Her2-high and Her2-low cell lines, compared to an immunoconjugate with a maytansine payload. An immunoconjugate with a non-Her2 antigen binder is included for comparison.

Fig. 16 shows in vivo tumor growth inhibition results on mouse xenograft tumors (SK-OV- 3ip) treated with trastuzumab alone, trastuzumab conjugated with compound 5B (5 mg/kg dose and 10 mg/kg dose), and a control conjugate where the antigen binding group does not recognize tumor antigens.

Fig. 17 shows in vivo tumor growth inhibition activity on mouse xenograft tumors

(SK_OV-3ip) treated with immunoconjugates having Eg5 inhibitor payloads on trastuzumab (anti-Her2 antibody) compared to conjugates with maytansine payloads and with one control lacking payload and one control without a tumor binding antibody.

Fig. 18 shows shows in vivo tumor growth inhibition activity on mouse xenograft tumors (H526) treated with immunoconjugates having Eg5 inhibitor payloads on cKitA (an anti- cKit antibody).

Fig. 19 shows in vivo tumor growth inhibition activity on mouse xenograft tumors (H526) treated with immunoconjugates having Eg5 inhibitor payloads on cKitA (an anti-cKit antibody) compared with a cKitA conjugate containing a maytansine payload (DM1 ).

Fig. 20 shows in vivo tumor growth inhibition activity on a mouse carrying two xenograft tumors (H526 and SK-OV-3ip) treated with immunoconjugates having Eg5 inhibitor payloads on a cKitA (an anti-cKit) antibody and on a trastuzumab antibody. Fig. 21 shows activity of a variety of Eg5 inhibitor immunoconjugates with trastuzumab antibody, tested on mouse xenograft SK-OV-3ip tumors. For comparison,

immunoconjugates having the same antibody conjugated with an auristatin payload (MMAE) and a maytansine payload (DM 1 ).

Fig. 22 shows in vivo activity of several immunoconjugates from Table 5 in mouse xenograft tumors, using Kadcyla® and the anti-Her2 antibody as comparators and vehicle-only as a control.

Fig. 23 shows in vivo activity of several immunoconjugates from Tables 5 in mouse xenografts, using Kadcyla®, and a Compound 6U— anti-Her2 antibody conjugate, and an igG 1 kappa chain specific for a viral glycoprotein, gH , as comparators and vehicle-only as a control.

Fig. 24 shows in vivo activity of several immunoconjugates from Tables 5 in mouse xenografts, using vehicle-only as a control. The immunoconjugates have anti-cKit antibodies conjugated to compounds from Table 5, and the cell line is sensitive to ckit antibodies.

Fig. 25 shows in vivo activity of several immunoconjugates from Tables 5 in mouse xenografts, using vehicle-only as a control. The immunoconjugates have anti-cKit antibodies conjugated to compounds from Table 5.

Fig. 26 shows in vivo activity of immunoconjugates having payload-linker Compound 5B in mouse xenografts, and three different anti-cKit antibodies. cKitA is the parent antibody; cKitB and cKitC are cysteine-engineered muteins of cKitA as described here. Results are shown at two different dosing levels, with vehicle-only as a control. At both doses, each of the cysteine-engineered mutant antibodies provided a more active immunoconjugate than the native antibody.

Fig. 27 shows in vivo activity of an immunoconjugate comprising the antibody cKitA conjugated with compound 5B, with the cKitA antibody (unconjugated) and vehicle as controls.

Fig. 28 includes graphs of in vitro (cell culture) activity of a wide range of

immunoconjugates of the invention, tested on a variety of different tumor cell types. The last figure (Figure 28-29) shows that in vitro activity is not significantly affected by small variations in DAR. DETAILED DESCRIPTION

The following definitions apply unless otherwise expressly provided.

The term "amino acid" refers to canonical, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the canonical amino acids. Canonical amino acids are proteinogenous amino acids encoded by the genetic code and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline serine, threonine, tryptophan, tyrosine, valine, as well as selenocysteine, pyrrolysine and pyrroline-carboxy-lysine. Amino acid analogs refer to compounds that have the same basic chemical structure as a canonical amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a canonical amino acid.

The term "antigen binding moiety" as used herein refers to a moiety capable of binding specifically to an antigen, and includes but is not limited to antibodies and antibody fragments.

The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region

(abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hyper variability, termed

complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. , effector cells) and the first component (Clq) of the classical complement system.

The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti- idiotypic (anti-Id) antibodies (including, e.g. , anti-Id antibodies to antibodies of the invention). The antibodies can be of any isotype/class (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g. , lgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2).

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (V_L) and heavy (V_H) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (C_L) and the heavy chain (CH 1 , CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino- terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and C_L domains actually comprise the carboxy-terminal domains of the heavy and light chain, respectively.

The term "antigen binding fragment", as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g. , by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH 1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al. , Nature 341 :544- 546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g. , Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879- 5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment." These antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v- NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1 126-1 136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Pat. No. 5,641 ,870).

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies and antigen binding fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term "human antibody", as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000).

The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing). The term "humanized" antibody, as used herein, refers to an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65- 92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991 ); Padlan, Molec. Immun., 31 (3):169-217 (1994).

The term "specifically binds" or "selectively binds," when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies, e.g., in a biological sample, e.g., a blood, serum, plasma or tissue sample. Thus, under certain designated immunoassay conditions, the antibodies or binding agents with a particular binding specificity bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. In one embodiment, under designated immunoassay conditions, the antibody or binding agents with a particular binding specificity bind to a particular antigen at least ten (10) times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require the antibody or agent to have been selected for its specificity for a particular protein. As desired or appropriate, this selection may be achieved by subtracting out antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some embodiments, antibodies or antibody fragments are selected that cross-react with certain desired molecules.

A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA

immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least than 10 to 100 times over the background. The term "affinity" as used herein refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody "arm" interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to canonical amino acid polymers as well as to non-canonical amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "immunoconjugate" or "antibody conjugate" as used herein refers to the linkage of an antigen binding moiety such as an antibody or an antigen binding fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an

immunotherapeutic agent, an imaging probe, a spectroscopic probe, and the like. The linkage can be covalent bonds, or non-covalent interactions, and can include chelation. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins (including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

The term "cytotoxin", or "cytotoxic agent" as used herein, refer to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.

The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents. The term "drug moiety" or "payload" as used herein, includes but is not limited to inhibitors of Eg5, refers to a chemical moiety that is or can be conjugated to the antibody or antigen binding fragment to form an immunoconjugate, and can include any moiety that is useful to attach to an antibody or antigen binding fragment. The immunoconjugates of the invention comprise an Eg5 inhibitor as a payload, for example, but may also include one or more other payloads. For example, a drug moiety or payload can be an anticancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent. In certain embodiments a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1 , a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Suitable examples include auristatins such as MMAE and MMAF;

calicheamycins such as gamma-calicheamycin; and maytansinoids such as DM1 , DM3 and DM4. Methods for attaching each of these to a linker compatible with the antibodies and method of the invention are known in the art. See, e.g., Singh et al., (2009)

Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457. In addition, a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide, a reactive functional group such as those described herein, or a binding agent that can connect the conjugate to another moiety or surface, etc.

"Tumor" refers to neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

The term "anti-tumor activity" means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. A possible way of showing anti-tumor activity is to show a decline in growth rate of abnormal cells that arises during therapy or tumor size stability or reduction. Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.

The term "malignancy" refers to a non-benign tumor or a cancer. As used herein, the term "cancer" includes a malignancy characterized by deregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.

The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

The term "a therapeutically effective amount" of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease.

In another non-limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reduce or inhibit the activity of Eg5. As used herein, the term "subject" refers to an animal. Typically the animal is a mammal. A subject also refers to for example, primates (e.g., humans, male or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In specific embodiments, the subject is a human.

As used herein, the term "inhibit", "inhibition" or "inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

As used herein, the term "treat", "treating" or "treatment" of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treat", "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treat", "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treat", "treating" or "treatment" refers to preventing or delaying progression of the disease or disorder.

As used herein, a subject is "in need of" a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

In certain embodiments, the modified immunoconjugates of the invention are described according to an "X group-to-antibody" ratio of, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8, or 12 or 16; this ratio corresponds to 'n' in Formula (I). While this ratio has an integer value for a specific conjugate molecule, it is understood that an average value is typically used to describe a sample containing many molecules, due to some degree of inhomogeneity within a sample of an immunoconjugate. The average loading for a sample of an immunoconjugate is referred to herein as the "drug to antibody ratio," or DAR. In some embodiments, the DAR is between about 1 and about 16, and typically is about 1 , 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least 50% of a sample by weight is compound having the average DAR plus or minus 2, and preferably at least 50% of the sample is a product that contains the average DAR plus or minus 1.5. Preferred embodiments include immunoconjugates wherein the DAR is about 2 to about 8, e.g., about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In these embodiments, a DAR of 'about q' means the measured value for DAR is within ±20% of q, or preferably within ±10% of q.

As used herein, the term "an optical isomer" or "a stereoisomer" refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers. It is understood that a substituent may be attached at a chiral center of a carbon atom. The term "chiral" refers to molecules which have the property of non-superimposability on their mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner. Therefore, the invention includes enantiomers, diastereomers or racemates of the compound. "Enantiomers" are a pair of stereoisomers that are non- superimposable mirror images of each other. A 1 :1 mixture of a pair of enantiomers is a "racemic" mixture. The term is used to designate a racemic mixture where appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror- images of each other. The absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.

Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible isomers or as mixtures thereof, for example as pure optical isomers, or as isomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. The present invention is meant to include all such possible isomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms, unless otherwise stated, e.g., where a specific isomer is identified. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a di-substituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.

As used herein, the terms "salt" or "salts" refers to an acid addition or base addition salt of a compound of the invention. "Salts" include in particular "pharmaceutical acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate,

bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorotheophyllinate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate,

methylsulphate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,

polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, tosylate and trifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like.

Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, use of non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where practicable. Lists of additional suitable salts can be found, e.g., in "Remington's

Pharmaceutical Sciences", 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹P, ³²P, ³⁵S, ³⁶CI, ¹²⁵l respectively. The invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as ³H and ¹⁴C, or those into which non-radioactive isotopes, such as ²H and ¹³C are present. Such isotopically labeled compounds are useful in metabolic studies (with ¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detection or imaging techniques, such as positron emission tomography (PET) or single- photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ¹⁸F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the

accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

Further, substitution with heavier isotopes, particularly deuterium (i.e., ²H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium

incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

The term 'thiol-maleimide' as used herein refers to a group formed by reaction of a thiol with maleimide, having this general formula

where Y and Z are groups to be connected via the thiol-maleimide linkage and can comprise linker components, antibodies or payloads.

'Cleavable' as used herein refers to a linking group or linker component that connects two moieties by covalent connections, but breaks down to sever the covalent connection between the moieties under physiologically relevant conditions, typically a cleavable linking group is severed in vivo more rapidly in an intracellular environment than when outside a cell, causing release of the payload to preferentially occur inside a targeted cell. Cleavage may be enzymatic or non-enzymatic, but generally releases a payload from an antibody without degrading the antibody. Cleavage may leave some portion of a linking group or linker component attached to the payload, or it may release the payload without any residue of the linking group.

'Pel' as used herein refers to pyrroline carboxy lysine, e.g.,

where R is H, which has the following formula when incorporated into a peptide:

The corresponding compound wherein R is methyl is pyrrolysine.

'Non-cleavable' as used herein refers to a linking group or linker component that is not especially susceptible to breaking down under physiological conditions, e.g., it is at least as stable as the antibody or antigen binding fragment portion of the

immunoconjugate. Such linking groups are sometimes referred to as 'stable', meaning they are sufficiently resistant to degradation to keep the payload connected to the antigen binding moiety Ab until Ab is itself at least partially degraded, i.e., the degradation of Ab precedes cleavage of the linking group in vivo. Degradation of the antibody portion of an ADC having a stable or non-cleavable linking group may leave some or all of the linking group, e.g., one or more amino acid groups from an antibody, attached to the payload or drug moiety that is delivered in vivo.

As used herein, the term "halogen" (or halo) refers to fluorine, bromine, chlorine or iodine, in particular fluorine or chlorine. Halogen-substituted groups and moieties, such as alkyl substituted by halogen (haloalkyl) can be mono-, poly- or per-halogenated.

As used herein, the term "hetero atoms" refers to nitrogen (N), oxygen (O) or sulfur (S) atoms, in particular nitrogen or oxygen, unless otherwise provided. As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon moiety. Unless otherwise provided, alkyl refers to hydrocarbon moieties having 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.

Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

A substituted alkyl is an alkyl group containing one or more substituents in place of hydrogen, such as one, two or three substituents, up to the number of hydrogens present on the unsubstituted alkyl group. Suitable substituents for alkyl groups, if not otherwise specified, may be selected from halogen, CN, oxo, hydroxy, Ci_₄ alkoxy, substituted or unsubstituted C₃.₆ cycloalkyl, substituted or unsubstituted phenyl, amino, (Ci-4 alkyl)amino, di(Ci_₄ alkyl)amino, Ci_ alkylthio, Ci_ alkylsulfonyl, -C(=0)- Ci_₄ alkyl, COOH, -COO(Ci_₄ alkyl), -0(C=0)- Ci_₄ alkyl, -NHC(=0) Ci_₄ alkyl and -NHC(=0)0 Ci_₄ alkyl groups. Preferred substituents for alkyl groups include halogen, CN, oxo, hydroxy, Ci_ alkoxy, C₃.₆ cycloalkyl, phenyl, amino, (Ci_ alkyl)amino, di(Ci_ alkyl)amino, Ci_ alkylthio, d_₄ alkylsulfonyl, -C(=0)- d_₄ alkyl, COOH, -COO(d_₄ alkyl), -0(C=0)- d_₄ alkyl, -NHC(=0) C-i-4 alkyl and -NHC(=0)0 Ci_ alkyl groups. In some embodiments, a Ci_ substituted alkyl has 1-3 substituents unless otherwise specified.

As used herein, the term "alkylene" refers to a divalent alkyl group having 1 to 10 carbon atoms, and two open valences to attach to other features. Unless otherwise provided, alkylene refers to moieties having 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to, methylene, ethylene, n-propylene, iso-propylene, n-butylene, sec-butylene, iso-butylene, tert-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2,2- dimethylpentylene, 2,3-dimethylpentylene, n-heptylene, n-octylene, n-nonylene, n- decylene and the like. A substituted alkylene is an alkylene group containing one or more, such as one, two or three substituents; unless otherwise specified, suitable substituents are selected from the substituents listed above for alkyl groups.

As used herein, the term "haloalkyl" refers to an alkyl as defined herein, which is substituted by one or more halo groups as defined herein. The haloalkyl can be monohaloalkyl, dihaloalkyl, trihaloalkyl, or polyhaloalkyl including perhaloalkyl. A monohaloalkyl can have one iodo, bromo, chloro or fluoro within the alkyl group. Chloro and fluoro are preferred on alkyl or cycloalkyl groups; fluoro, chloro and bromo are often preferred on aryl or heteroaryl groups. DihaloalkyI and polyhaloalkyi groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyi. Typically the polyhaloalkyi contains up to 12, or 10, or 8, or 6, or 4, or 3, or 2 halo groups. Non-limiting examples of haloalkyi include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A perhalo-alkyl refers to an alkyi having all hydrogen atoms replaced with halo atoms, e.g, trifluoromethyl.

As used herein, the term "alkoxy" refers to alkyl-O-, wherein alkyi is defined above. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. Typically, alkoxy groups have 1 -10, or 1 -6 carbons, more commonly 1 -4 carbon atoms.

A "substituted alkoxy" is an alkoxy group containing one or more, such as one, two or three substituents on the alkyi portion of the alkoxy. Unless otherwise specified, suitable substituents are selected from the substituents listed above for alkyi groups, except that hydroxyl and amino are not normally present on the carbon that is directly attached to the oxygen of the substituted 'alkyl-O' group.

Similarly, each alkyi part of other groups like "alkylaminocarbonyl", "alkoxyalkyl", "alkoxycarbonyl", "alkoxy-carbonylalkyl", "alkylsulfonyl", "alkylsulfoxyl", "alkylamino", shall have the same meaning as described in the above-mentioned definition of "alkyi". When used in this way, unless otherwise indicated, the alkyi group is often a 1 -4 carbon alkyi and is not further substituted by groups other than the component named. When such alkyi groups are substituted, suitable substituents are those named above for alkyi groups unless otherwise specified.

As used herein, the term "haloalkoxy" refers to haloalkyl-O-, wherein haloalkyi is defined above. Representative examples of haloalkoxy include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, trichloromethoxy, 2-chloroethoxy, 2,2,2- trifluoroethoxy, 1 , 1 , 1 ,3,3,3-hexafluoro-2-propoxy, and the like. Typically, haloalkyi groups have 1 -4 carbon atoms.

As used herein, the term "cycloalkyl" refers to saturated or unsaturated non- aromatic monocyclic, bicyclic, tricyclic or spirocyclic hydrocarbon groups of 3-12 carbon atoms: the cycloalkyl group may be unsaturated, and may be fused to another ring that can be saturated, unsaturated or aromatic, provided the ring atom of the cycloalkyl group that is connected to the molecular formula of interest is not an aromatic ring carbon.

Unless otherwise provided, cycloalkyi refers to cyclic hydrocarbon groups having between 3 and 9 ring carbon atoms or between 3 and 7 ring carbon atoms. Preferably, cycloalkyi groups are saturated monocyclic rings having 3-7 ring atoms unless otherwise specified.

A substituted cycloalkyi is a cycloalkyi group substituted by one, or two, or three, or more than three substituents, up to the number of hydrogens on the unsubstituted group. Typically, a substituted cycloalkyi will have 1 -4 or 1 -2 substituents. Suitable substituents, unless otherwise specified, are independently selected from the group consisting of halogen, hydroxyl, thiol, cyano, nitro, oxo, Ci.₄-alkylimino, Ci_₄-alkoximino, hydroxyimino, Ci_₄-alkyl, C₂-4-alkenyl, C₂-4-alkynyl, Ci_₄-alkoxy, C₁_₄-thioalkyl, C₂_4- alkenyloxy, C2-4-alkynyloxy, Ci-₄ alkylcarbonyl, carboxy, Ci-4-alkoxycarbonyl, amino, C1-4- alkylamino, di- Ci_₄-alkylamino, Ci_₄-alkylaminocarbonyl, di- Ci_₄-alkylaminocarbonyl, C1-4- alkylcarbonylamino, Ci-4-alkylcarbonyl(Ci-₄-alkyl)amino, Ci_₄-alkylsulfonyl, Ci_

4alkylsulfamoyl, and Ci.₄ alkylaminosulfonyl, where each of the aforementioned

hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more groups independently selected at each occurrence from the list of preferred substituents for 'alkyl' groups herein. Preferred substituents for a cycloalkyi include Ci_₄ alkyl, halogen, CN, oxo, hydroxy, Ci_₄ alkoxy, amino, (Ci_₄ alkyl)amino, di(Ci-₄ alkyl)amino, Ci_₄ alkylthio, Ci-₄ alkylsulfonyl, -C(=0)- C1-4 alkyl, COOH, -COO(Ci_4 alkyl), -0(C=0)- Ci_4 alkyl, -NHC(=0) Ci_4 alkyl and -NHC(=0)0 Ci_4 alkyl groups.

Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl and the like. Exemplary bicyclic hydrocarbon groups include bornyl, indyl, hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1 .1 ]hexyl, bicyclo[2.2.1 ]heptyl, bicyclo[2.2.1 Jheptenyl, 6,6-dimethylbicyclo[3.1 .1 Jheptyl, 2,6,6-trimethylbicyclo[3.1 .1 Jheptyl, bicyclo[2.2.2]octyl and the like. Exemplary tricyclic hydrocarbon groups include adamantyl and the like.

Similarly, each cycloalkyi part of other groups like "cycloalkyloxy",

"cycloalkoxyalkyl", "cycloalkoxycarbonyl", "cycloalkoxy-carbonylalkyl", "cycloalkylsulfonyl", "halocycloalkyl" shall have the same meaning as described in the above-mentioned definition of "cycloalkyi". When used in these terms, the cycloalkyi is typically a monocyclic 3-7 carbon ring that is unsubstituted or substituted with 1 -2 groups. When optionally substituted, the substituents are typically selected from C1-C4 alkyl and those set forth above as suitable for cycloalkyl groups.

As used herein, the term "aryl" refers to an aromatic hydrocarbon group having 6- 14 carbon atoms in the ring portion. Typically, aryl is monocyclic, bicyclic or tricyclic aryl having 6-14 carbon atoms, often 6-10 carbon atoms, e.g., phenyl or naphthyl.

Furthermore, the term "aryl" as used herein, refers to an aromatic substituent which can be a single aromatic ring, or multiple aromatic rings that are fused together. Non-limiting examples include phenyl, naphthyl and 1 ,2,3,4-tetrahydronaphthyl, provided the tetrahydronaphthyl is connected to the formula being described through a carbon of the aromatic ring of the tetrahydronaphthyl group.

A substituted aryl is an aryl group substituted by 1-5 (such as one, or two, or three) substituents independently selected from the group consisting of hydroxyl, thiol, cyano, nitro, C-i-4-alkyl, C₂-4-alkenyl, C₂-4-alkynyl, Ci_₄ alkoxy, Ci_₄-thioalkyl, C₂-4-alkenyloxy, C2-4- alkynyloxy, halogen, C Ci-4-alkylcarbonyl, carboxy, Ci-4-alkoxycarbonyl, amino, C1-4- alkylamino, di- Ci_₄-alkylamino, Ci_₄ alkylaminocarbonyl, di- Ci_₄-alkylaminocarbonyl, C1-4- alkylcarbonylamino, Ci-₄ alkylcarbonyl(Ci-₄ alkyl)amino, Ci_₄ alkylsulfonyl, sulfamoyl, Ci_₄ alkylsulfamoyl, and Ci.₄ alkylaminosulfonyl where each of the afore-mentioned

hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more groups independently selected at each occurrence from the groups listed above as preferred substituents for alkyl groups. Preferred substituents for aryl groups are Ci_₄ alkyl, halogen, CN, hydroxy, Ci_₄ alkoxy, amino, (Ci_₄ alkyl)amino, di(Ci_₄ alkyl)amino, Ci_₄ alkylthio, Ci_₄ alkylsulfonyl, -C(=0)- Ci_₄ alkyl, COOH, -COO(Ci_₄ alkyl), -0(C=0)- alkyl, -NHC(=0) d.₄ alkyl and -NHC(=0)0 d.₄ alkyl groups.

Similarly, each aryl part of other groups like "aryloxy", "aryloxyalkyl",

"aryloxycarbonyl", "aryloxy-carbonylalkyl" shall have the same meaning as described in the above-mentioned definition of "aryl".

As used herein, the term "heterocyclyl" refers to a heterocyclic radical that is saturated or partially unsaturated but not aromatic, and can be a monocyclic or a polycyclic ring (in case of a polycyclic ring particularly a bicyclic, tricyclic or spirocyclic ring); and has 3 to 14, more commonly 4 to 10, and most preferably 5 or 6 ring atoms; wherein one or more, preferably one to four, especially one or two ring atoms are heteroatoms independently selected from O, S and N (the remaining ring atoms therefore being carbon). Even though described as, e.g., a C5-6 atom ring, a heterocycle contains at least one heteroatom as a ring atom and has the number of ring atoms stated, e.g. 5-6 in this example. Preferably, a heterocyclyl group has one or two such heteroatoms as ring atoms, and preferably the heteroatoms are not directly connected to each other. The bonding ring (i.e. the ring connecting to the Formula of interest) preferably has 4 to 12, especially 5 to 7 ring atoms. The heterocyclic group can be fused to an aromatic ring, provided the atom of the heterocyclic group attached to the Formula of interest is not aromatic. The heterocyclic group can be attached to the Formula of interest via a heteroatom (typically nitrogen) or a carbon atom of the heterocyclic group. The heterocyclyl can include fused or bridged rings as well as spirocyclic rings, and only one ring of a polycyclic heterocyclic group needs to contain a heteroatom as a ring atom. Examples of heterocycles include tetrahydrofuran (THF), dihydrofuran, 1 ,4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3-dioxolane, imidazolidine, imidazoline, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3-dithiane, oxathiane, thiomorpholine, and the like.

A substituted heterocyclyl is a heterocyclic group independently substituted by 1-5 (such as one, or two, or three) substituents selected from the substituents described above for a cycloalkyl group.

Similarly, each heterocyclyl part of other groups like "heterocyclyloxy",

"heterocyclyloxyalkyl", "heterocyclyloxycarbonyl" shall have the same meaning as described in the above-mentioned definition of "heterocyclyl".

"Cyclic ether" as used herein refers to a heterocyclic ring containing 4-7 ring atoms unless otherwise specified, which contains an oxygen atom as a ring member, and optionally two non-adjacent oxygen atoms for rings of five or more atoms. Typical examples include oxetane, tetrahydrofuran, tetrahydropyran, oxepane, and 1 ,4-dioxane.

As used herein, the term "heteroaryl" refers to a 5-14 membered monocyclic- or bicyclic- or tricyclic-aromatic ring system, having 1 to 8 heteroatoms as ring members; the heteroatoms are selected from N, O and S. Heteroaryl and heterocyclic rings may be referred to herein as, e.g., C₅.₆ heteroaryl or heterocyclic: it is understood when this description is used that 5-6 refers to the total number of ring atoms, including both carbon and heteroatoms; such rings may alternatively be referred to as 5-6 membered heteroaryl or heterocyclic groups. Typically, the heteroaryl is a 5-10 membered ring system, e.g., a

5-6 membered monocyclic or an 8-10 membered bicyclic group containing at least one heteroatom as a ring member. Typical heteroaryl groups include 2- or 3-thienyl, 2- or 3- furyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-imidazolyl, 1-, 3-, 4-, or 5- pyrazolyl, 2-, 4-, or 5-thiazolyl,

3- , 4-, or 5-isothiazolyl, 2-, 4-, or 5-oxazolyl, 3-, 4-, or 5-isoxazolyl, 3- or 5-1 ,2,4-triazolyl,

4- or 5-1 ,2, 3-triazolyl, 1 - or 2-tetrazolyl, 2-, 3-, or 4-pyridyl, 3- or 4-pyridazinyl, 3-, 4-, or 5- pyrazinyl, 2-pyrazinyl, and 2-, 4-, or 5-pyrimidinyl.

The term "heteroaryl" also refers to a group in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment to the Formula of interest is on a heteroaromatic ring. Nonlimiting examples include 1-, 2-, 3-, 5-, 6-, 7-, or 8- indolizinyl, 1-, 3-, 4-, 5-, 6-, or 7-isoindolyl, 2-, 3-, 4-, 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-indazolyl, 2-, 4-, 5-, 6-, 7-, or 8- purinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, or 9-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinoliyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8- isoquinoliyl, 1-, 4-, 5-, 6-, 7-, or 8-phthalazinyl, 2-, 3-, 4-, 5-, or 6-naphthyridinyl, 2-, 3- , 5-,

6- , 7-, or 8-quinazolinyl, 3-, 4-, 5-, 6-, 7-, or 8-cinnolinyl, 2-, 4-, 6-, or 7-pteridinyl, 1-, 2-, 3-,

4- , 5-, 6-, 7-, or 8-4aH carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-carbzaolyl, 1-, 3-, 4-, 5-, 6-,

7- , 8-, or 9-carbolinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenanthridinyl, 1- , 2-, 3-, 4-, 5-, 6-, 7-, 8-, or 9-acridinyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9-perimidinyl, 2-, 3-, 4-, 5-, 6-, 8-, 9-, or 10- phenathrolinyl, 1-, 2- , 3-, 4-, 6-, 7-, 8-, or 9-phenazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10- phenothiazinyl, 1-, 2-, 3-, 4-, 6-, 7-, 8-, 9-, or 10-phenoxazinyl, 2-, 3-, 4-, 5-, 6-, or I-, 3-, 4-,

5- , 6-, 7-, 8-, 9-, or 10- benzoisoquinolinyl, 2-, 3-, 4-, or thieno[2,3-b]furanyl, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10 -, or 1 1-7H-pyrazino[2,3-c]carbazolyl,2-, 3-, 5-, 6-, or 7-2H- furo[3,2-b]- pyranyl, 2-, 3-, 4-, 5-, 7-, or 8-5H-pyrido[2,3-d]-o-oxazinyl, 1-, 3-, or 5-1 H-pyrazolo[4,3-d]- oxazolyl, 2-, 4-, or 54H-imidazo[4,5-d] thiazolyl, 3-, 5-, or 8-pyrazino[2,3-d]pyridazinyl, 2-,

3- , 5-, or 6- imidazo[2,1-b] thiazolyl, 1-, 3-, 6-, 7-, 8-, or 9-furo[3,4-c]cinnolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 8-, 9-, 10, or 1 1-4H-pyrido[2,3-c]carbazolyl, 2-, 3-, 6-, or 7-imidazo[1 ,2- b][1 ,2,4]triazinyl, 7-benzo[b]thienyl, 2-, 4-, 5- , 6-, or 7-benzoxazolyl, 2-, 4-, 5-, 6-, or 7- benzimidazolyl, 2-, 4-, 4-, 5-, 6-, or 7-benzothiazolyl, 1-, 2-, 4-, 5-, 6-, 7-, 8-, or 9- benzoxapinyl, 2-, 4-, 5-, 6-, 7-, or 8-benzoxazinyl, 1-, 2-, 3-, 5-, 6-, 7-, 8-, 9-, 10-, or 11- 1 H-pyrrolo[1 ,2-b][2]benzazapinyl. Typical fused heteroaryl groups include, but are not limited to 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolinyl, 2-, 3-,

4- , 5-, 6-, or 7-indolyl, 2-, 3-, 4-, 5-, 6-, or 7-benzo[b]thienyl, 2-, 4-, 5- , 6-, or 7- benzoxazolyl, 2-, 4-, 5-, 6-, or 7-benzimidazolyl, and 2-, 4-, 5-, 6-, or 7-benzothiazolyl.

A substituted heteroaryl is a heteroaryl group containing one or more substituents selected from the substituents described above as suitable for an aryl group. Similarly, each heteroaryl part of other groups like "heteroaryloxy",

"heteroaryloxyalkyl", "heteroaryloxycarbonyl" shall have the same meaning as described in the above-mentioned definition of "heteroaryl".

In one aspect, the invention provides immunoconjugates (e.g., ADCs) that comprise an inhibitor of Eg5 as the drug or payload, and compositions and methods using such immunoconjugates or ADCs to treat cell proliferation disorders. Certain imidazole and triazole compounds are known in the art as inhibitors of Eg5 and as therapeutic agents to treat cell proliferation disorders, and can be used as ADC payloads; see for example WO2007/021794, WO2006/002236, WO2008/063912, WO2009/077448, WO201 1/128381 , and WO201 1/128388. Other Eg5 inhibitors known in the art that could be adapted for use as ADC payloads include, for example, compounds disclosed in WO2006/049835, U.S. Patent No. 7,504,405, U.S. Patent No. 7,939,539, and in Figure 3 of Rath and Kozielski, Nature Reviews: Cancer, vol. 12, 527-39 (Aug. 2012).

Immunoconjugates that comprise an Eg5 inhibitor as payload (drug) include conjugates of Formula (I):

(I)

L represents a linking group that connects X to Ab by covalent or non-covalent bonding, which may optionally attach more than one X to Ab, and which may or may not contain a linker component that is cleavable;

X represents an Eg5 inhibitor, such as a compound of Formula (II) or Formula (III) as described herein, or other inhibitors of Eg5 including compounds disclosed in Rath (Rath and Kozielski, Nature Rev. Cancer, vol. 12, 527-39 (2012)), including ispinesib, SB- 743921 , AZD4877, ARQ621 , ARRY-520, LY2523355, MK-0731 , EMD534085, and GSK- 923295, and Eg5 inhibitors described in WO06/002236, WO2007/021794,

WO2008/063912, WO2009/077448, WO201 1/128381 , WO201 1/128388, and

WO2006/049835; m is an integer from 1-4, typically 1-2; and n is an integer from 1 to 16, preferably 2-8.

Certain aspects and examples of the invention are provided in the following listing of enumerated embodiments:

1. An immunoconjugate of Formula (I):

(I)

wherein Ab represents an antigen binding moiety;

L represents a linking group that connects X to Ab;

m is an integer from 1-4;

n is an integer from 1 to 16; and

X represents a group of Formula (II)

that is connected by L to Ab, wherein: Z is N or CH;

Ar¹ is phenyl optionally substituted with up to three groups selected from halo, C1-3 alkyl, and C1.3 haloalkyl;

Ar² is phenyl or pyridinyl or a 4-6 atom cyclic ether, and Ar² is optionally substituted with up to two groups selected from halo, CN, C1.3 alkyl, hydroxyl, amino, and C1-3 haloalkyl; R¹ is C-i-6 alkyl, -(CH₂)o-2-C₃-6 cycloalkyl, or -(CH₂)o-2-C₄-7 heterocyclyl (a 4-7 membered heterocycle) containing up to two heteroatoms selected from N, O and S as ring members, wherein each Ci_₆ alkyl, C₃.₆ cycloalkyl, or C₄.₇ heterocyclyl is optionally substituted with up to three groups selected from halo, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, hydroxyl, amino, oxo, hydroxyl-substituted Ci_ alkyl, amino-substituted C1.4 alkyl, Ci_ alkyl-amino, and COO(Ci_ alkyl); and further optionally including -C(0)-Ci_₆ alkyl, -C(O)- NH-d.6 alkyl, and -C(0)0-Ci_-6 alkyl;

R² is H or Ci-4 alkyl;

T is (CH₂)₁.₃;

Y is selected from C1.3 aminoalkyl, C .₆ heterocyclyl, and C₃.₆ cycloalkyl, wherein C1-3 aminoalkyl, C₄.₆ heterocyclyl, and C₃.₆ cycloalkyl are each optionally substituted with up to two groups selected from amino, oxo, halo, hydroxyl, Ci_ alkyl, Ci_ alkoxy, hydroxyl- substituted C1-4 alkyl, amino-substituted C1-4 alkyl, COOH, COO-(Ci-₄ alkyl),

alkyl), alkyl)₂, and C1-4 haloalkyl;

A is NH, N(Ci- alkyl), or a bond between the carbonyl in Formula (II) and Q;

Q is selected from Ci_₄ alkyl, -0-Ci_₄alkyl, -(CH₂)₀-₂-C₄.₆heterocyclyl, -(CH₂)₀.₂-C₃. ecycloalkyl, -(CH₂)₀-₂-C₅-₆heteroaryl, and -(CH₂)₀.₂-phenyl, and is optionally substituted with up to three groups selected from halo, hydroxyl, amino, -SH, -R, -OR, -SR, -S0₂R, -NHR, -O-glucuronate, and -NR₂, where each R is Ci_₆ alkyl optionally substituted with halo, -SH, -NH₂, OMe, or -OH; in some embodiments, R can also be a C₃-e cycloalkyl, or a 4-6 membered heterocycle containing N, O or S as a ring member, and each R is

independently optionally substituted with halo, -SH, -NH₂, OMe, or -OH.

Typically m is 1 or 2, preferably 1 ; and n is 2-8, preferably about 2 to about 4, or between 3 and 5.

In some embodiments, n is 2, 4, 6 or 8. In some embodiments, where more than one L is present, each L is independently selected. In other embodiments, each L is the same.

In some of these embodiments, R¹ is Ci_-6 alkyl, -(CH₂)₀-₂-C₃.₆ cycloalkyl, or -(CH₂)₀.

₂-C₄-7 heterocyclyl containing up to two heteroatoms selected from N, O and S as ring members, wherein each Ci_-6 alkyl, C₃.₆ cycloalkyl, or C₄.₇ heterocyclyl is optionally substituted with up to three groups selected from halo, Ci_ alkyl, Ci_ haloalkyl, Ci_ alkoxy, hydroxyl, amino, oxo, hydroxyl-substituted Ci_₄ alkyl, Ci_₄ alkyl-amino, and COO(Ci_₄ alkyl). In certain embodiments, each C1-6 alkyl, C3-6 cycloalkyl, or C4-7 heterocyclyl is substituted with up to two groups selected from halo, Ci_₄ alkyl, C1.4 haloalkyl, Ci_₄ alkoxy, hydroxyl, amino, and hydroxyl-substituted Ci_₄ alkyl, with preferred substituents selected from F, hydroxy, methoxy, and amino. Examples of suitable R¹ groups include t-butyl, 2-

methoxy-2-propyl, 4-tetrahydropyranyl, and groups of the formula

wherein A is -OH, -NH₂, -COOH, -CONH₂, -NHC(0)H, or -SH; and in some of these embodiments, linker L is attached to moiety A, by replacement of one of the hydrogen atoms of R¹ or A.

Certain embodiments of R¹ include 4-tetrahydropyranyl and

and optionally also wherein A is -OH, -NH₂, -COOH, -CONH₂, -NHC(0)H, or -SH, and the dashed line indicates the point of attachment of each R¹ to Formula II.

1 A. In some of the foregoing embodiments, R² is H.

I B. In some of the foregoing embodiments, A is a bond between the carbonyl and Q. In other embodiments, A is NH.In some of the foregoing embodiments, T is CH₂ when Y is a heterocyclyl or cycloalkyl, and T is CH₂ or CH₂CH₂ when Y is Ci_₃ aminoalkyl.

I C. In some of the foregoing embodiments, Q is Ci_ alkyl substituted with one or two groups selected from hydroxy, amino, thiol, amino-Ci_₄-alkyloxy or amino-Ci_₄- alkylthio. In other embodiments, Q is a ring selected from morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, piperazine, phenyl and pyridine, where the ring is optionally substituted with up to two groups selected from Ci_₄ alkyl, halo, CN, hydroxy, amino, Ci_ alkyl-amino, Ci_ alkylsulfonyl, and Ci_ alkoxy.

I D. In some of the foregoing embodiments, Y is a pyrrolidine ring optionally substituted with up to two groups selected from halo, Ci_ alkyl, hydroxy, amino, hydroxy- Ci_4 alkyl, amino-Ci_ alkyl, Ci_ alkyl-amino, and Ci_ alkoxy. Preferred substituents for the pyrrolidine include F, methyl, hydroxy, and hydroxymethyl. 2. The immunoconjugate according to embodiment 1 , wherein R² is H.

3. The immunoconjugate according to embodiment 1 or embodiment 2, wherein Z is CH.

4. The immunoconjugate according to embodiment 1 or 2, wherein Z is N.

5. The immunoconjugate according to any one of embodiments 1 to 4, wherein R¹ is a tetrahydropyranyl ring, and R¹ is optionally substituted with up to two groups selected from oxo and methyl.

6. The immunoconjugate according to any of the preceding embodiments, wherein Ar¹ is dihalophenyl. In certain embodiments, Ar¹ is 2,5-dihalophenyl, e.g., Ar¹ can be 2,5- difluorophenyl.

In these embodiments, Ar² can be phenyl, halophenyl, hydroxyphenyl, or aminopyridine e.g., phenyl, 3-fluorophenyl, 3-hydroxyphenyl, 3-amino-2-pyridinyl.

7. The immunoconjugate according to any of the preceding embodiments, wherein the compound of Formula (II) has the formula:

wherein L in Formula (I) is attached to Y, or to Q, or to R¹ in Formula (II). In preferred embodiments, L is attached to an oxygen atom or amine nitrogen that is part of group Y or part of group Q.

7A. The immunoconjugate according to any of the preceding embodiments, wherein the compound of Formula (II) has the formula:

wherein L is attached to R¹ , and R¹ is an optionally substituted alkyl group. In some of these embodiments, R¹ is a C₃-e alkyl group of general formula -CMe₂(CH₂)o-2-G-[L], where [L] indicates the point where R¹ is attached to L, and G can be a bond, -0-, -NH-, - S-, -CONH-or -COO-. In some of these embodiments, R¹ is -C(Me)₂-(CH₂)o-2-R³⁰, wherein R³⁰ is hydroxy, carboxy, or amino. In these embodiments, L is often attached to R¹ via the group R³⁰. Embodiments of R¹ include -C(Me)₂-(CH₂)₀-2-O-[L], -C(Me)₂-(CH₂)₀- 2-NH-[L], -C(Me)₂-(CH₂)o-₂-C(=0)-[L], and -C(Me)₂-(CH₂)₀-₂-C(=O)-NH-[L], where [L] indicates the point at which the compound of Formula (II) is attached to linker L in Formula (I).

8. The immunoconjugate according to any of embodiments 1 -7, wherein R¹ is 4-

tetrahydropyranyl. For example, R¹ is . Optionally, the tetrahydropyran ring can be substituted by one or two substituents selected from hydroxy, methyl, methoxy, and halo.

9. The immunoconjugate according to any of the preceding embodiments, wherein Q in Formula (II) is Ci_₄ alkyl substituted with one or two groups selected from hydroxyl and amino. In embodiments where A is NH or N(alkyl), Q is often -CH₂OH, -CH₂NH₂, or C₂_4 alkyl, substituted with one or two groups selected from -OH and -NH₂. Where A is a bond, Q can be Ci_₃ alkyl, optionally substituted with -OH and/or NH₂. A hydroxyl or amine of group Q can be used to attach the compound of Formula (II) to L in Formula (I).

10. The immunoconjugate according to any of the preceding embodiments, wherein Y is pyrrolidine optionally substituted with one or two groups selected from fluoro, amino, hydroxyl, methoxy, and hydroxymethyl. In these embodiments, the pyrrolidine ring NH, or an amino or hydroxyl on the pyrrolidine ring, can be the point of attachment of the compound of Formula (II) to L in Formula (I).

1 1 . The immunoconjugate according to any of the preceding embodiments, wherein A is -NH-.

1 1 B. Alternatively, the immunoconjugate of any of the preceding embodiments wherein A is a bond.

12. The immunoconjugate of any of the preceding embodiments, wherein the linking group is cleavable. Cleavable linking groups include a linker component such as a dipeptide that provides a site for enzymatic cleavage in cells (e.g., val-cit); a linker component such as a hydrazone or imine that is pH sensitive and prone to cleavage inside cells; a disulfide linker component that tends to cleave inside cells; or a

glucuronidase-sensitive linker component such as a p-aminobenzyloxycarbonyl moiety having an-O-glucuronic acid group on the phenyl ring of the aminobenzyloxy group.

13. The immunoconjugate of any of embodiments 1 -1 1 , wherein the linking group is non-cleavable.

13A. The immunoconjugate of embodiment 13, wherein the linker is substituted with a polar group selected from -(CH₂)i_-2-COOH, -(CH₂)i-₂-OH, -COOH or -S0₃H, or a pharmaceutically acceptable salt thereof.

14. A compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

Z is N or CH; Ar¹ is phenyl optionally substituted with up to three groups selected from halo, Ci_₃ alkyl, and Ci_₃ haloalkyl;

Ar² is phenyl or pyridinyl, and is optionally substituted with up to two groups selected from halo, CN, Ci_₃ alkyl, hydroxyl, amino, and Ci_₃ haloalkyl;

R¹ is -(CH₂)o-2-C₄-7 heterocyclyl or -(CH₂)o-2-C₃.₇ cycloalkyl, where the C₄-₇ heterocyclyl is a 4-7 membered ring containing up to two heteroatoms selected from N, O and S as ring members, and C₄.₇ heterocyclyl and C₃.₇ cycloalkyl are each optionally substituted with up to three groups selected from halo, Ci_₄ alkyl (e.g. , methyl), Ci_ haloalkyl (e.g. , trifluoromethyl), Ci_ alkoxy, hydroxyl, amino, oxo, hydroxyl-substituted Ci_ alkyl, amino-substituted Ci_ alkyl, or COO(Ci_ alkyl);is optionally substituted with up to three groups selected from halo, d-4 alkyl, d-4 alkoxy, oxo, or -COO(d-4 alkyl);

R² is H or d-4 alkyl;

T is (CH₂)₁.₃;

Y is selected from Ci_₂ aminoalkyl, C .₆ heterocyclyl, and C₃.₆ cycloalkyl, wherein C1-2 aminoalkyl, C₄.₆ heterocyclyl, and C₃.₆ cycloalkyl are each optionally substituted with up to two groups selected from amino, oxo, halo, hydroxyl, Ci_ alkoxy, hydroxyl- substituted Ci-4 alkyl, amino-substituted d-4 alkyl, COOH, COO-(d-4 alkyl), and d-3 haloalkyl;

A is NH, N(d_ alkyl), or a bond between the carbonyl in Formula (I I I) and Q;

Q is selected from d-4 alkyl, -(CH₂)₀-₂-d-₆heterocyclyl, -(CH₂)₀-₂-C₅-₆heteroaryl, and -(CH₂)₀-₂-phenyl, and Q is optionally substituted with up to three groups selected from halo, hydroxyl, amino, -SH, -R, -OR, -SR, -S0₂R, -N₃, -NHR, -O-glucuronate, and -NR₂, where each R is Ci_₆ alkyl optionally substituted with halo, -SH, -NH₂, OMe, or -OH.

In some of these embodiments of Formula (I I I), R¹ is -(CH₂)₀.₂-C₃-6 cycloalkyl, or - (CH₂)o-₂-C₄-7 heterocyclyl containing up to two heteroatoms selected from N, O and S as ring members, wherein each C₃.₆ cycloalkyl, or C -7 heterocyclyl is optionally substituted with up to three groups selected from halo, C₁.₄ alkyl, d-4 haloalkyl, d-4 alkoxy, hydroxyl, amino, oxo, hydroxyl-substituted d-4 alkyl, d-4 alkyl-amino, and COO(d_ alkyl). In certain embodiments, d-e cycloalkyl, or C4-7 heterocyclyl is substituted with up to two groups selected from halo, d-4 alkyl, d-4 haloalkyl, d-4 alkoxy, hydroxyl, amino, and hydroxyl-substituted Ci_₄ alkyl, with preferred substituents selected from F, hydroxy, methoxy, and amino.

In certain embodiments, R¹ is selected from 4-tetrahydropyranyl and

and optionally also where wherein A is -OH, -NH₂, -COOH, -CONH₂, -NHC(0)H, or -SH and the dashed line indicates the point of attachment for each R¹.

In some of the foregoing embodiments of Formula (III), R² is H. In some of the foregoing embodiments of Formula (I I I), A is a bond between the carbonyl and Q. In other embodiments, A is NH. In some of the foregoing embodiments, T is CH₂ when Y is a heterocyclyl or cycloalkyi, and T is CH₂ or CH₂CH₂ when Y is Ci_₃ aminoalkyl.

In some of the foregoing embodiments of Formula (I I I), Q is Ci_₄ alkyl substituted with one or two groups selected from hydroxy, amino, thiol, amino-Ci_₄-alkyloxy or amino- Ci-₄-alkylthio. In other embodiments, Q is a ring selected from morpholine,

thiomorpholine, pyrrolidine, tetrahydrofuran, piperazine, phenyl and pyridine, where the ring is optionally substituted with up to two groups selected from Ci_ alkyl, halo, CN, hydroxy, amino, Ci_₄ alkyl-amino, Ci_₄ alkylsulfonyl, and Ci_ alkoxy.

In some of the foregoing embodiments of Formula (I I I), Y is a pyrrolidine ring optionally substituted with up to two groups selected from halo, Ci_ alkyl, hydroxy, amino, hydroxy-Ci_ alkyl, amino-Ci_ alkyl, Ci_ alkyl-amino, and Ci_ alkoxy. Preferred substituents for the pyrrolidine include F, methyl, hydroxy, and hydroxymethyl.

These novel Eg5 inhibitors can be used to treat cancer as low-molecular weight drug compounds, or they can be incorporated into an ADC for targeted in vivo delivery.

15. The compound of embodiment 14, wherein R¹ is tetrahydropyranyl; in some embodiments R¹ is tetrahydropyran-4-yl.

16. A compound of Formula ( 11 A) or (MB)

(MA) (MB) wherein Ar¹, Ar², Z, R¹ , R², T, Q, Y, and A are as defined for Formula (II) in embodiment 1 above,

Q^* is selected from -CH₂-, -CH(Me)-, -CH(Me)CH₂-, -CH₂CH₂-, -CH₂0-, -CH₂S-, - CH₂-NH-, -CH₂-NMe-, -CH(Me)0-, -CH(OH)-CH₂0-, -CH(0-)-CH₂OH, -CH(OH)-CH₂NH-, - CH(NH-)-CH₂OH, -CH(0-)-CH₂NH₂, -CH(NH-)-CH₂OH, -CH(Me)S-, -CH(Me)NH- , -CH₂CH₂0-, -CH₂CH₂NH-, -CH₂CH₂S-, -CH(Me)CH₂0-, -CH(Me)CH₂S- , -CH(Me)CH₂NH-,

and in some of these embodiments, Q^* is selected from -CH₂0-, -CH₂S-, - CH₂-NH-, -CH₂-NMe-, -CH(Me)0-, -CH(OH)-CH₂0-, -CH(0-)-CH₂OH, - CH(OH)-CH₂NH-, -CH(NH-)-CH₂OH, -CH(0-)-CH₂NH₂, -CH(NH-)- CH₂OH, -CH(Me)S-, -CH(Me)NH-, -CH₂CH₂0-, -CH₂CH₂NH-, -CH₂CH₂S-, - CH(Me)CH₂0-, -CH(Me)CH₂S-, -CH(Me)CH₂NH-,

, and

Y* is selected from -CH(CH₂F)NH-, -CH₂NH-,

ρίί^1" H ^H

, and where R¹⁰ and R¹¹ are independently H, Me, OMe, F, CH₂F, CH₂OH, COOH, COO(d_₄ alkyl), CONH(d_₄ alkyl), CON(C₁_₄ alkyl)₂, or OH; and W is a linking moiety that comprises one or more linker components and a reactive functional group. Suitable linking moieties with reactive functional groups such as maleimide are disclosed herein, including

42

43

R^a = H, R = CH₂Ph

nyl)

R^a = H, R^b = Ph

Ra = H, R^b = 3-indolyl

 and optionally also including

where X represents the compound of Formula (IIA) or (MB), and LG is a leaving group suitable to provide an acylating agent, such as CI, -O-Benzotriazole (-OBt), -O- Azabenzotriazole (-OAt), -O-succinimide, substituted phenoxy, -OC(0)(phenyl or substituted phenyl), -OC(0)(Ci_-6 alkyl), or -OC(0)0(Ci_-6 alkyl).

16B. An alternative embodiment includes a compound of Formula (IIC):

wherein Ar¹, Ar², Z, R², T, Q, Y, W, and A are as defined for Formula (MA) and (MB) above, R is C3-6 alkyl optionally substituted with oxo, hydroxy, amino, or carboxy, e.g., R is -C(Me)₂-(CH₂)o-2-A, wherein A is amino, hydroxy, carboxy, CONH₂, or -SH; and W is a linking moiety that comprises one or more linker components and a reactive functional group.

For example, W can be -L¹-L²-L³-L⁴-L⁵-G, wherein G is the reactive functional group, and L¹, L², L³, L⁴ and L⁵ are linker components selected from those described herein. Suitable reactive functional groups (G) are ones having suitable reactivity to form a covalent linkage to an amino acid side chain of an amino acid in an antibody or antigen binding moiety, such as -SH or -NH₂ of a cysteine or lysine, respectively. Examples of suitable reactive functional groups include maleimide, alpha-halo acetamides (halo = CI, Br or I), aldehyde (CHO), thiol (to form disulfides), 2-aminobenzaldehydes (ABA), 2- amino-benzophenones (ABP), 2-aminoacetophenones (AAP), carboxylates, and activated esters that readily form amides with free amine groups, such as esters of N- hydroxysuccinimide and its analogs. Suitable reactive functional groups ABA, AAP and ABP include the following groups:

(ABA) (AAP) (ABP)

These moieties, placed at the end of an optional linking group opposite the payload, react with Pel or Pyl as described in Ou, et al., Proc. Nat'l Acad. Sci. 2011 , 108(26), 10437-42 to form a linking group where L¹ is

or wherein Fc^u is H or Me, and R is H, Me or Phenyl.

These embodiments of the invention are activated intermediates useful for the preparation of conjugates comprising an Eg5 inhibitor payload similar to the compounds of Formula (II) and (III) described above. In these embodiments, the compounds comprise a reactive functional group positioned at a location that is well tolerated, even for use with non-cleavable linkers, e.g., the linking group attaches to an atom

corresponding to Y or Q in Formula (II).

17. The compound of embodiment 16, wherein W comprises a reactive functional group selected from -SH, -NH₂, -C(=0)H, -C(=0)Me, N-maleimide, -NHC(=0)-CH₂-halo, - COOH, and -C(=0)-OR', wherein halo is selected from CI, Br and I, and -OR' is the leaving group moiety of an activated ester.

18. The compound of any of embodiments 14-17, wherein Ar¹ is dihalophenyl.

Particularly suitable groups include 2,5-difluorophenyl, 2-Fluoro-5-chlorophenyl and 2- chloro-5-fluorophenyl.

19. The compound of any of embodiments 14-18, wherein Ar² is phenyl or halophenyl. Particularly suitable groups include phenyl and 3-fluorophenyl.

20. The compound of any of embodiments 14-19, wherein Z is CH.

21. The compound of any of embodiments 14-19, wherein Z is N.

22. The compound of any of embodiments 16-21 , wherein R¹ is 4-tetrahydropyranyl.

22A. The compound of any of embodiments 16-21 , wherein R¹* is -C(Me)₂CH₂C(0)NH- [W], where [W] indicates the point of attachment of R^1* to W.

23. The compound of any of embodiments 14-22, wherein R² is H. In alternative embodiments, R² can be methyl.

24. The compound of any of embodiments 14-23, wherein A is -NH-.

25. The compound of any of embodiments 14-23, wherein A is a bond.

26. The compound of any of embodiments 14-25, wherein T is CH₂ or CH₂CH₂. Preferably, T is CH₂CH₂ when Y or Y* is an aminoalkyl such as -CH(CH₂F)NH₂ or - CH₂NH₂; and T is -CH₂- when Y or Y^* is an optionally substituted pyrrolidine, such as

, or

27. The compound of any of embodiments 14-26, wherein Y is selected

from -CH(CH₂F)NH₂, , and where R¹⁰ and R¹¹ are independently H, Me, OMe, F, CH₂F, CH₂OH, COOH, COO(d_₄ alkyl), or OH.

In certain embodiments of such compounds, Y is selected from -CH(CH₂F)NH₂,

, and

Preferred embodiments of Y include

where [T] indicates the point of attachment of Y to T in the formula.

Preferred embodiments of Y^* include

where [T] indicates the point of attachment of Y^* to T in the formula; and [W] indicates where Y^* attaches to W.

28. The compound of any of embodiments 14-27, wherein Q is selected from - CH₂OH, -CH₂-NH₂, -CH(Me)OH, -CH(OH)-CH₂OH, -CH(OH)-CH₂NH₂, -CH(NH₂)-CH₂OH, -CH(NH₂)-CH₂OH, -CH(Me)SH, -CH(Me)NH₂, -CH₂CH₂OH, -CH₂CH₂NH₂, -CH₂CH₂SH, - CH(Me)CH₂OH, -CH(Me)CH₂SH, -CH(Me)CH₂NH₂,

, and

Preferred embodiments of the combination -A-Q include -CH₂OH, -CH(Me)OH, -NH-CH₂-CHOH-CH₂OH, -NH-CH₂-CH₂OH, and -NH-CHMe-CH₂OH. In particular, -A-Q can be selected from

where [CO] indicates the point of attachment of -A-Q to the carbonyl in the

Formula. 29. The compound of embodiment 14, which is selected from the compounds in Table 1 and the pharmaceutically acceptable salts thereof.

30. A pharmaceutical composition comprising a compound of any of embodiments 15- 29 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

31 . A combination comprising a therapeutically effective amount of a compound according to one of embodiments 14-15 or a pharmaceutically acceptable salt thereof and one or more therapeutically active co-agents.

32. A method of treating a cell proliferation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate of any of embodiments 1 -13, or a compound of any of embodiments 14-15, or a

pharmaceutically acceptable salt thereof.

33. A compound according to any one of embodiments 14-15 or an immunoconjugate of any of embodiments 1 -13 or a pharmaceutically acceptable salt thereof, for use as a medicament.

34. The compound according to embodiment 33 or a pharmaceutically acceptable salt thereof, wherein the medicament is for use in the treatment of cancer.

In certain embodiments, the cancer is selected from gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, renal, gastric (e.g., gastrointestinal stromal tumors (GIST)), liver, and pancreatic cancer, and sarcoma.

35. An immunoconjugate of any of embodiments 1 -13 or a pharmaceutically acceptable salt thereof, for use to treat cancer.

36. An immunoconjugate according to embodiment 1 , having a formula selected from:

R^a = H, R = CH₂Ph

nyl)

R^a = H, R^b = 3-indolyl

where each R^N is independently H or CH₂CH₂-R³⁰, where R³⁰ is hydroxy, amino or carboxy, and R^N is preferably H;

54

55

wherein R ^o is H or Me, and R^0U is H, Me or Phenyl. and optionally also

Particular examples include these:

and optionally also

37. An immunoconjugate of any of embodiments 1-14 or a pharmaceutically acceptable salt thereof, for use to treat cancer. 38. An immunoconjugate Ab-L^*-X, comprising a payload (X) linked to an antibody (Ab), wherein the linking group L^* comprises a group of the formula -C(0)NR²¹- or -NR²¹-C(0)- wherein R²¹ is of the formula -(CH₂)i-4-R²², where R²² is a polar group selected from -OH, -IMH2, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH20)k-OCH2CH₂OR²³, and -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci_₄ alkyl. Preferably, X is an inhibitor of Eg5, including a compound of any of embodiments 14-29.

39. An immunoconjugate of Formula (I):

(I)

wherein Ab represents an antigen binding moiety;

L represents a linking group that connects X to Ab;

m is an integer from 1-4;

n is an integer from 1 to 16; and

X independently at each occurrence represents an inhibitor of Eg5. In many embodiments, X is a compound according to any of embodiments 14-29.

In certain of these embodiments, X is a compound of this formula:

wherein R^4a is H, F or OH;

R is H or F; R is selected from

Y is selected from

and Q is selected from

where linker L is attached to X at Y⁴, Q⁴, or R¹.

Preferred linkers L for these embodiments include, where [Ab] designates the point of attachment to the antibody:

combine to form this group -Q⁴-L:

40. The immunoconjugate of embodiment 39, wherein X is a compound selected from Table 1.

41. The immunoconjugate of embodiment 39 or 40, wherein m is 1 and the immunoconjugate is formed by reaction of Ab with a compound selected from Table 2.

42. An immunoconjugate made by reaction of an antibody containing at least one free thiol group with a maleimide compound selected from the following group:

61

In some embodiments of these immunoconjugates, the antibody is selected from anti- estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti- HER-2 antibody, anti-cKit antibody, anti-EGFR antibody, anti-cathepsin D antibody, andti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti- CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti- Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD1- antibody, anti-CD1 1c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA/CD45 antibody, anti-CD45RO antibody, anti- CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chains antibody, anti- melanosomes antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody, and anti-Tn-antigen antibody.

These immunoconjugates may have a drug to antibody ratio (DAR) between 1 and 8, typically between 2 and 6, and preferably between 3 and 5.

44. An immunoconjugate of any one of embodiments 1-14 or 38-43, wherein the antigen binding moiety is an antibody or antigen binding fragment thereof having at least one non-native cysteine residue introduced into the constant region, where the linking group L is attached to the non-native cysteine residue.45. The immunoconjugate of claim 44, wherein m is 1 and n is between 1 and 5, preferably about 2 or about 4.

46. The immunoconjugate of embodiment 44 wherein said antibody or antigen binding fragment thereof comprises a combination of substitution of two or more amino acids with non-native cysteine on its constant regions.

47. The immunoconjugate of embodiment 46, wherein the non-native cysteine substitutions are selected from position 360 of an antibody heavy chain, and position 107 of an antibody kappa light chain, wherein said positions are numbered according to the EU system.

48. The immunoconjugate of embodiment 46 wherein the non-native cysteine are selected from positions 152 and 375 of an antibody heavy chain, wherein said positions are numbered according to the EU system.

In the above enumerated embodiments, Ab can be any antigen binding moiety unless otherwise defined, and is preferably an antibody or antigen binding fragment that recognizes a cell surface marker such as those described herein that is characteristic of a targeted cell, such as a cancer cell. A tumor-associated antigen is especially suitable. In the enumerated embodiments, X can be any compound of Formula (I I) or (II I), particularly any of the compounds disclosed in embodiments 1 -1 1 above or in embodiments 14-15, and including any of the species in Table 1 . In preferred implementations of embodiment 36, X is selected from:

where [L] indicates which atom of X is attached to the linking group shown in embodiment 36.

In additional embodiments, X is of the formula:

Ab in any of the above embodiments, unless otherwise described, can be any antigen binding moiety, typically one that recognizes an antigen characteristic of cells to be targeted for pharmaceutical intervention, such as cancer cells. Many suitable antigens are well known in the art; specific ones of special interest are described herein. Typically, Ab is an antibody, which may be isolated or constructed, and may be natural or modified (engineered), or an antibody fragment that retains antigen binding activity similar to the antibody.

L in the above embodiments can be any linking group that connects Ab to one or more X groups, including a single bond directly connecting Ab to an atom of a compound of Formula (II). Suitable linkers for use in ADCs are well known in the art, and can be used in the conjugates of the invention. L can be attached to Ab at any suitable available position on Ab: typically, L is attached to an available amino nitrogen atom (i.e., a primary or secondary amine, rather than an amide) or a hydroxylic oxygen atom, or to an available sulfhydryl, such as on a cysteine.

In some of these embodiments of Formula (I), m is 1 or 2, and m is preferably 1. In some of these embodiments of Formula (I), n is 1-10, commonly 1-8 or 1-6, and preferably n is 1 , 2, 3, 4, or 5.

In some embodiments of the compounds of Formula (II), 11 A, MB, and III, R¹ is or comprises a 3-6 membered cycloalkyi ring or a 4-6 membered heterocyclic group, and may be substituted as described in the various enumerated embodiments. In some embodiments, R¹ is a 5-6 membered heterocyclic group that is unsubstituted. In other embodiments, R¹ is a 5-6 membered heterocyclic group substituted by an amine or hydroxyl, either of which is optionally a point of attachment for the linking group.

In any of the foregoing embodiments, L can be comprised of up to six linker components, L¹ , L², L³, L⁴, L⁵ and L⁶, as further described herein. Thus for example, the immunoconjugate of Formula (I) can be of the Formula (IA):

(IA) wherein Ab represents an antigen binding moiety;

L¹, L², L³, L⁴, L⁵, and L⁶ each independently represent a linker component; n is an integer from 1 to 16; and

X represents an Eg5 inhibitor, e.g., a compound of Formula (II) or Formula (III) as described herein.

These immunoconjugates can equivalently be depicted as follows, to indicate that the linker component L⁶ is attached to the compound of Formula (II):

wherein Ab represents an antigen binding moiety;

L¹, L², L³, L⁴, L⁵, and L⁶ each independently represent a linker component; n is an integer from 1 to 16; and Ar¹, Ar², R¹, R², T, Y, A, Q and Z are as defined for Formula (II) or Formula (III) herein. L⁶ in this formula is attached to the chemical structure shown: -L⁶- can be considered a substituent of the group of Formula (II) or (III). In some embodiments L⁶ is attached to an atom of Q, Y, or R¹, often at an oxygen atom or nitrogen atom of Q, Y or R¹ or one of their substituents.

In these embodiments, each linker component can optionally be a bond joining the groups on either side of the linker component, so in some embodiments the compounds of Formula (I A) include 0, 1 , 2, 3, 4, 5, or 6 of the linker components L¹, L², L³, L⁴, L⁵, and L⁶ connecting Ab to X.

Suitable linker components for forming linking group L are known in the art, as are methods for constructing the linking group L. These components include the groups commonly used to attach a group to an amino acid, spacers such as alkylene groups and ethylene oxide oligomers, amino acids and short peptides up to about 4 amino acids in length; a bond; and carbonyl, carbamate, carbonate, urea, ester and amide linkages, and the like.

In some embodiments of these conjugates, L¹ is selected from groups formed upon reaction of a reactive functional group with one of the amino acid side chains commonly used for conjugation, e.g., the thiol of cysteine, or the free -NH₂ of lysine, or a Pel or Pyl group engineered into an antibody. See e.g., Ou, et al., PNAS 108(26), 10437- 42 (201 1 ). Suitable -L¹- groups include, but are not limited to, a single bond,

particularly for attaching to a cysteine residue of Ab; and

particularly for attaching to the -NH₂ of a lysine residue of Ab, where each p is 1 -10, and each R is independently H or Ci_₄ alkyl (preferably methyl); and

or

wherein R is H or Me, and R is H, Me or Phenyl, for linking to a Pel or Pyl group, where the acyl group shown attaches to the lysine portion of a Pel or Pyl in an engineered antibody.

Suitable options for linker components L², L³, L⁴, and L⁵ include, for example, alkylene groups -(CH₂)_n- (where n is typically 1-10 or 1-6), ethylene glycol units (- CH₂CH₂0-)n (where n is 1-20, typically 1-10 or 1-6), -0-, -S-, carbonyl (-C(=0)-), amides - C(=0)-NH- or -NH-C(=0)- , amides or carbamates comprising -C(=0)-NR²¹- or -NR²¹- C(=0)- where R²¹ is a substituted alkyl with a polar substituent as described herein, esters -C(=0)-0- or -0-C(=0)-, ring systems having two available points of attachment such as a divalent ring selected from phenyl (including 1 ,2- 1 ,3- and 1 ,4- di-substituted phenyls), C₅-6 heteroaryl, C₃-₈ cycloalkyl including 1 ,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and 1 ,4-disubstituted cyclohexyl, and C₄.₈ heterocyclyl rings, and specific examples depicted below; amino acids -NH-CHR^*-C=0- or -C(=0)-CHR^*- NH-, or groups derived from amino acids that attach to N of an adjacent structure (e.g., to a maleimide nitrogen) having the formula [N]-CHR^*-C(=0)- where R^* is the side chain of a known amino acid (frequently one of the canonical amino acids, e.g., trp, ala, asp, lys, gly, and the like, but also including e.g. norvaline, norleucine, homoserine, homocysteine, phenylglycine, citrulline, and other commonly named alpha-amino acids), polypeptides of known amino acids (e.g., dipeptides, tripeptides, tetrapeptides, etc.), thiol-maleimide linkages (from addition of -SH to maleimide), -S-CR₂- and other thiol ethers such as -S- CR₂-C(=0)- or -C(=0)-CR₂-S- where R is independently at each occurrence H or Ci_₄ alkyl, -CH₂-C(=0)-, and disulfides (-S-S-), as well as combinations of any of these with other linker components described below, e.g., a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer. In some embodiments, each linker component L¹ , L², L³, L⁴, L⁵, and L⁶ is selected from the group consisting of a bond, -(CH₂)_q-, -(CR₂)_q-, -(CH₂CH₂0)_q-, -(CH₂)_q-NR-(CH₂)_q- , -(CH₂)_q-0-(CH₂)_q-, -NH-CHR*-C(=0)-, -C(=0)-CHR*-NR-, -CHR*-C(=0)-, -C(=0)NR- , -NRC(=0)-, -C(=0)0-, -OC(=0)-, -NRC(=0)0-, -OC(=0)NR-, -(CH₂)_qS(CH₂)_q-,

72

wherein R²⁰ is H or Me, and R³⁰ is H, Me or Phenyl, where each q is 0-10, preferably 0-6 or 1-6; each R, R⁵, and R⁶ is independently H or Ci-₄ alkyl,

R^* is a side chain of a common amino acid such as gly, ala, trp tyr, phe, leu, val, asp, glu gin, asn, his, arg, lys, cys, met, ser, thr, phenylglycine, t-butylglycine;

R⁷ is independently selected from H, Ci_₄ alkyl, phenyl, pyrimidine and

pyridine;

R is independently selected from

, and ;

R⁹ is independently selected from H, Ci_₄ alkyl, and Ci_₆ haloalkyl; and any or all of L¹ to L⁶ can be absent, i.e., any or all of them can represent a bond between the two groups to which they are attached.

In certain embodiments, the linker L comprises at least one linker component of the formula -C(0)-NR²¹- or -NR²¹-C(0)-, or -0-C(=0)-NR²¹-, -NR²¹-C(=0)-0-, wherein R²¹ is of the formula -(CH₂)i-₄-R²², where R²² is a polar group such as -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k-OCH₂CH₂OR²³, or -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci_₄ alkyl. It has been found that use of this moiety in a linking group connecting an antibody to a payload reduces aggregation of the conjugate, and thereby improves the solubility and efficacy of the immunoconjugate. In preferred embodiments, R²² is -(CH₂)₂-OH or -(CH₂)₂-COOH.

Particularly suitable options for linker component L⁶ include a covalent bond as explained herein, carbonyl [ -C(=0)- ],

where G is an enzyme-cleavable group such as glucuronate, each q is 1 -10, Z is a polar group such as -COOH or -S0₃H, and each R is independently H or Ci_₄ alkyl (preferably H or methyl).

In another aspect, the invention provides an immunoconjugate Ab-L*-X, comprising a payload (X) linked to an antibody (Ab), wherein the linking group L^* comprises a group of the formula -C(0)NR²¹- or -NR²¹-C(0)- wherein R²¹ is of the formula -(CH₂)i-₄-R²², where R²² is a polar group selected from -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k-OCH₂CH₂OR²³, and -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci_-4 alkyl. The payload can be any suitable payload, such as a cytotoxin like a maytansinoid, auristatin, amatoxin or amanitin, or other known payloads having therapeutic utility in an ADC. In some embodiments of these

immunoconjugates, X is an Eg5 inhibitor such as those as described herein.

X in embodiments of Formula I can be any Eg5 inhibitor, but is preferably a compound of Formula II as described above, or any of the sub-classes of this Formula that are described in the enumerated embodiments, such as a compound of Formula (III) as described above. In some embodiments, X is a compound selected from Table 1. While Formula (II) and (III) describe 'neutral' compounds, it is understood that in the context of the conjugates, X comprises one atom that is covalently attached to L or directly to Ab.

Unless otherwise provided, X is attached to the linking group in the above formulas via any available position. In some embodiments, X is attached to the linking group via one of the atoms of the group represented by Q, or the group represented by Y, or the group represented by R¹ in either Formula (II) or Formula (III).

Similarly, Ab can be any antigen binding moiety, including those described herein. Preferably, Ab is an antibody, which may be modified; e.g., Ab can have other payloads attached in addition to at least one Eg5 inhibitor of the present invention. In embodiments where Ab is attached to a succinimide ring or to a -CH₂- or -S- of the linking group L, it is typically connected via a sulfur atom of a cysteine of Ab; in embodiments where Ab is attached to the linking group at a carbonyl of the linking group, it is typically attached via a nitrogen atom, such as the amine of a lysine, in Ab.

The invention contemplates use of any small-molecule Eg5 inhibitor as a cytotoxic payload for immunoconjugates. It is illustrated with Eg5 inhibitors of Formula (II), but is not limited to these inhibitors, and has been demonstrated to work with other classes of Eg5 inhibitors. In preferred embodiments, the Eg5 inhibitor is a compound of Formula (II) or (III), particularly including any of the compounds in Table 1.

It is understood that compounds of Formula (II) or (III), when they are part of an immunoconjugate, are covalently attached to a linking group L (or to a linker component that is part of L), or to Ab itself. Thus, in the immunoconjugates of the invention, the compounds of Formula (II) or (III) have an open valence whereby they are linked covalently to L (or directly linked to Ab), preferably tightly enough for in vivo delivery to cells targeted for inhibition or elimination. Typically, the link between the Eg5 inhibitor and Ab involves covalent connection of the antigen binding moiety Ab to the Eg5 inhibitor(s), often through a linking group comprising one or more linker components, such as those described herein.

In use, either before or, more typically, after an ADC reaches and binds to an antigen on a targeted cell, the Eg5 inhibitor will be released from Ab: preferably, the Eg5 inhibitor is released primarily within the targeted cell, after the ADC binds to a surface antigen and is then internalized into the targeted cell. In some embodiments, the linking group L is designed to be cleavable, and the Eg5 inhibitor detaches from the ADC following internalization.

In some embodiments, the linking group is not designed to be cleavable, and release of the Eg5 inhibitor results when the antigen binding group (e.g., antibody) is degraded in vivo. Typically, degradation of Ab occurs inside a targeted cell, as by protease digestion. In these embodiments, at least a portion of linking group L may remain attached to the Eg5 inhibitor X, provided the portion of linking group L that remains on X does not interfere with sub-micromolar affinity of the inhibitor X for inhibition of Eg5.

A wide variety of linking groups for use in ADCs are known (see, e.g., Lash, Antibody-Drug Conjugates: the Next Generation of Moving Parts, Start-Up, Dec. 201 1 , 1 - 6), and can be used in conjugates within the scope of the invention. A linking group can be a single covalent bond between an atom of the Eg5 inhibitor and an atom of the antibody; for example, Q can be an alkyl group such as methyl and A can be absent in Formula (II), providing an Eg5 inhibitor of this formula:

To attach this inhibitor to an antigen binding moiety, it can be converted into a modified Eg5 inhibitor of the following formula, having an iodide (I) as a reactive functional group:

The iodide compound, an alpha-halo acetamide, can react directly with a free thiol group on an antibody, providing an immunoconjugate of this formula:

wherein S is the sulfur atom of a cysteine residue of the antibody, and the linking group L in Formula (I) represents the covalent bond between CH₂ and S.

In other embodiments of the conjugates of Formula (I), L can be comprised of two, three, four, five, six, or more than six linker components, e.g., L¹ , L², L³, L⁴, L⁵, and L⁶. Many linkers comprising multiple linker components are known in the art, and the various linker components can be selected and combined to provide operable immunoconjugates of the invention. In certain embodiments, the immunoconjugate is of the Formula (IA):

(IA) wherein Ab represents an antigen binding moiety;

L¹ , L², L³, L⁴, L⁵, and L⁶ represent linker components;

n is an integer from 1 to 16; and X represents an Eg5 inhibitor, e.g., a compound of Formula (II) or Formula (III) as described herein.

In such compounds, L¹ is typically selected from groups formed upon reaction of a reactive functional group with one of the amino acid side chains commonly used for conjugation, e.g., the thiol of cysteine, or the free -NH₂ of lysine on an antibody, or a Pel or Pyl group engineered into an antibody. See e.g., Ou, et al., PNAS 108(26), 10437-42 (2011 ). Suitable -L¹- groups include, but are not limited to, a single bond as described above,

particularly for attaching to a cysteine residue of Ab; and

particularly for attaching to a lysine residue of Ab, where each n is 1-10, and each R is independently H or Ci_₄ alkyl (preferably methyl).

Suitable options for linker components L², L³, L⁴, and L⁵ include, for example, in addition to a bond, alkylene groups -(CH₂)_n- (where n is typically 1-10 or 1-6), ethylene glycol units (-CH₂CH₂0-)n (where n is typically 1-10 or 1-6), -0-, -S-, carbonyl (-C(=0)-), amides -C(=0)-NH- or -NH-C(=0)-, esters -C(=0)-0- or -0-C(=0)-, rings having two available points of attachment such as divalent phenyl, C₅.₆ heteroaryl, C₃.₈ cycloalkyl or C₄-8 heterocyclyl groups, amino acids -NH-CHR^*-C=0- or -C(=0)-CHR^*-NH-, or groups derived from amino acids that attach to N (e.g., to a maleimide nitrogen) having the formula [N]-CHR^*-C(=0)- , where R^* is the side chain of a known amino acid (frequently one of the canonical amino acids, but also including e.g. norvaline, norleucine, homoserine, homocysteine, phenylglycine, citrulline, and other named alpha-amino acids), polypeptides of known amino acids (e.g., dipeptides, tripeptides, tetrapeptides, etc.), thiol- maleimide linkages (from addition of -SH to maleimide), -S-CR₂- and other thiol ethers such as -S-CR₂-C(=0)- or -C(=0)-CR₂-S-,where R is independently at each occurrence H or C1-4 alkyl, -CH₂-C(=0)-, and disulfides (-S-S-), as well as combinations of any of these with other linker components described below, e.g. , a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a self-immolative spacer.

In some embodiments, each linker component is selected from the group consisting of a bond between the groups on either side (meaning the linker component is effectively absent, so that the groups flanking it are connected together), -(CH₂)_q-, - (CH₂CH₂0)_q-, -(CH₂)_q-NR-(CH₂)_q-, -NH-CHR*-C(=0)-, -CHR*-C(=0)-, -C(=0)-CHR*-NH-, -C(=0)NH-, -NHC(=0)-, -C(=0)0-, -OC(=0)-, -NHC(=0)0-, -OC(=0)NH-, -(CH₂)_qS(CH₂)_q-

81

where each q is 0-10, preferably 0-6 or 1-6; each R, R⁵, and R⁶ is independently H or C^ alkyl,

R⁷ is independently selected from H, Ci_₄ alkyl, phenyl, pyrimidine and pyridine;

R⁸ is independently selected from

, and

R is independently selected from H, Ci_₄ alkyl, and Ci_₆ haloalkyl; and each R^* represents the side chain of an amino acid, which can be one of the amino acids encoded by the genetic code, or an alpha-amino acid analog such as citrulline, t-butyl glycine, phenyl glycine, homoserine, and the like; and any or all of these can be absent, i.e., they can represent a bond between the two groups to which they are attached.

Preferred options for linker component L⁶ include a covalent bond, carbonyl [

C(=0)- ],

where G is an enzyme-cleavable group such as glucuronate, each n is 1 -10, and each R is independently H or Ci_₄ alkyl (preferably methyl).

Another aspect of the invention provides linkers that reduce ADC aggregation and thus improve ADC function and properties. It is well known that aggregation of ADCs can be detrimental to their activity, and that aggregation depends on the characteristics of the payload as well as the linker. Certain hydrophilic linkers have been used to reduce aggregation. Example 4 illustrates novel linkers (e.g., linkers in ADC-1 1 1 and ADC-1 12) that reduce aggregation. These novel linkers comprise a linker component that is an N- substituted amide or carbamate of general formula -C(0)-NR²¹- or -NR²¹-C(0)- or -O-

C(=0)-NR²¹- or -NR²¹-C(=0)-0-, where R²¹ is an alkyl group substituted with a polar group such as hydroxy, amino, mono- or di-alkyl amine, carboxylate, carboxamide, or alkyl sulfonyl. As the data in Example 4 and Figures 10(A)-10(C) demonstrate, adding a polar group on the amide of a simple linker reduces aggregation of an ADC that otherwise exhibits significant aggregation as measured by size exclusion chromatography. Thus the invention includes ADCs of the general formula Ab-L^*-X wherein Ab is an antibody such as those described herein, X is a payload such as a cytotoxin or an Eg5 inhibitor such as any of the Eg5 inhibitors described herein, and L^* is a linker that comprises an amide of the formula -C(0)NR - or -NR -C(O)- or a carbamate of the formula -O- C(=0)-NR²¹- or -NR²¹-C(=0)-0 wherein R²¹ is of the formula -(CH₂)i-₄-R²², where R²² is a polar group selected from -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k- OCH₂CH₂OR²³, and -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci-₄ alkyl. Preferred embodiments of the linker comprise those wherein R²¹ is of the formula - (CH₂)₁.₂R²³; preferred embodiments of R²³ include hydroxy and carboxy.

Likewise, many antigens associated with cancer cells are known, and antibodies that bind to these antigens can be used in immunoconjugates within the scope of the invention. For example, while the clinical candidate ADCs reported in Lash utilize only four payload classes, they include at least 15 antigens associated with various targeted cells. Representative examples of the immunoconjugates of the invention are described herein, but the examples do not limit the scope of the invention or the claims.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

Various enumerated embodiments of the invention are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.

Pharmaceutically acceptable solvates in accordance with the invention include those wherein the solvent of crystallization may be isotopically substituted, e.g. D₂0, d⁶- acetone, d⁶-DMSO, as well as solvates with non-enriched solvents.

Compounds of the invention, i.e. compounds of formula (I) that contain groups capable of acting as donors and/or acceptors for hydrogen bonds may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals may be prepared from compounds of formula (I) by known co-crystal forming procedures. Such procedures include grinding, heating, co-subliming, co-melting, or contacting in solution compounds of formula (I) with the co-crystal former under crystallization conditions and isolating co- crystals thereby formed. Suitable co-crystal formers include those described in WO 2004/078163. Hence the invention further provides co-crystals comprising a compound of formula (I). Any asymmetric atom (e.g., carbon or the like) of the compound(s) of the present invention can be present in racemic or enantiomerically enriched, for example the (R)-, (S)- or (R,S)- configuration. In certain embodiments, each asymmetric atom has at least 50 % enantiomeric excess, at least 60 % enantiomeric excess, at least 70 % enantiomeric excess, at least 80 % enantiomeric excess, at least 90 % enantiomeric excess, at least 95 % enantiomeric excess, or at least 99 % enantiomeric excess of either the (R)- or (S)- configuration; i.e., for optically active compounds, it is often preferred to use one enantiomer to the substantial exclusion of the other enantiomer. Substituents at atoms with unsaturated double bonds may, if possible, be present in cis- (Z)- or trans- (E)- form.

Accordingly, as used herein a compound of the present invention can be in the form of one of the possible isomers, rotamers, atropisomers, tautomers or mixtures thereof, for example, as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (antipodes), racemates or mixtures thereof. 'Substantially pure' or 'substantially free of other isomers' as used herein means the product contains less than 5%, and preferably less than 2%, of other isomers relative to the amount of the preferred isomer, by weight.

Any resulting mixtures of isomers can be separated on the basis of the

physicochemical differences of the constituents, into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and/or fractional crystallization.

Any resulting racemates of final products or intermediates can be resolved into the optical antipodes by known methods, e.g., by separation of the diastereomeric salts thereof, obtained with an optically active acid or base, and liberating the optically active acidic or basic compound. In particular, a basic moiety may thus be employed to resolve the compounds of the present invention into their optical antipodes, e.g., by fractional crystallization of a salt formed with an optically active acid, e.g., tartaric acid, dibenzoyl tartaric acid, diacetyl tartaric acid, di-0,0'-p-toluoyl tartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. Racemic products can also be resolved by chiral chromatography, e.g., high pressure liquid chromatography (HPLC) using a chiral adsorbent.

Furthermore, the compounds of the present invention, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization. The compounds of the present invention may inherently or by design form solvates with pharmaceutically acceptable solvents (including water); therefore, it is intended that the invention embrace both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, and the like. The term "hydrate" refers to the complex where the solvent molecule is water.

The compounds of the present invention, including salts, hydrates and solvates thereof, may inherently or by design form polymorphs.

Eq5 Inhibitors

The ADCs of the invention can include any suitable Eg5 inhibitor, especially inhibitors having a molecular weight under 1000 Da, preferably under 700 Da. In some embodiments, the Eg5 inhibitor has an IC-50 less than 1 micromolar; in preferred embodiments, an Eg5 inhibitor for use as a payload has an IC-50 less than 100 nanomolar (nM). IC50's for this purpose can be measured as described in

WO2006/002236. Suitable Eg5 inhibitors include compounds disclosed in Rath (Rath and Kozielski, Nature Rev. Cancer, vol. 12, 527-39 (2012), including ispinesib, SB- 743921 , AZD4877, ARQ621 , ARRY-520, LY2523355, MK-0731 , EMD534085, and GSK- 923295, and Eg5 inhibitors described in WO06/002236, WO2007/021794,

WO2008/063912, WO2009/077448, WO201 1/128381 , WO201 1/128388, and

WO2006/049835: preferred payloads are compounds of Formula (II) and (III) described herein.

MK-0731 SB-743921 ispinesib Eg5 inhibitors of Formula (II) or (III) for use as ADC payloads can be attached to linking group L (or directly to Ab) at various positions on the inhibitor; in some

embodiments, a compound of Formula (II) is attached to L via an atom of group Q or Y or R¹. Any available valence on the compound of Formula (II) can be attached to L, but for convenient preparation of the conjugate or of modified Eg5 inhibitors of Formula (IIA) or (MB), attachment to L typically occurs at a heteroatom (N, O or S) of Q or Y. In some embodiments of the conjugates of Formula (I), the compound of Formula (II) comprises a free -NH- or free -OH or free -SH, which is used to attach the compound of Formula (II) to linking group L. In some embodiments, the free -NH-, -OH, or -SH is a portion of group Q or Y or R¹ in Formula (II). Note that the free -NH- can be an amino group (-NH₂), cyclic amine (e.g., -NH- in a cyclic group such as pyrrolidone, piperidine, or morpholine), or a secondary acyclic amine; in each case, the -NH- group is preferably not part of an amide or conjugated to a carbonyl or to an aryl or heteroaryl ring, which would reduce its reactivity.

Methods of attaching such payloads to a linking group for constructing conjugates are known in the art. Commonly, a free primary or secondary amine or a hydroxyl group is conjugated by an acylation reaction, using a linker component that comprises an activated ester, such as an N-hydroxysuccinimide ester or sulfonate-substituted N- hydroxysuccinimide ester to form an ester or amide linkage. Alternatively, a primary amine can be conjugated by formation of a Schiff base with a carbonyl (typically -CH(=0) or -C(=0)Me) of a linker component. Where the Eg5 inhibitor comprises a thiol group, the conjugate can be formed with a linker component comprising a maleimide or an alpha-halo acetamide (-NH-C(=0)-CH₂LG where LG is Br, CI or I), or it can be conjugated to a thiol-containing linker component or antigen binding moiety by forming a disulfide linkage.

In one aspect of the invention, an Eg5 inhibitor of Formula (III) is provided. The compounds of Formula III may be used as small-molecule therapeutic agents, or they may be incorporated as a payload in an ADC.

(III)

or a pharmaceutically acceptable salt thereof, wherein:

Z is N or CH;

Ar¹ is phenyl optionally substituted with up to three groups selected from halo, Ci_₃ alkyl, and Ci_₃ haloalkyl;

Ar² is phenyl or pyridinyl, optionally substituted with up to two groups selected from halo, CN, Ci_₃ alkyl, hydroxyl, amino, and Ci_₃ haloalkyl;

R¹ is -(CH₂)o-2-C₄-7 heterocyclyl, where the C₄-₇ heterocyclyl contains up to two heteroatoms selected from N, O and S as ring members and is optionally substituted with up to three groups selected from halo, d-4 alkoxy, hydroxyl, amino, oxo, hydroxyl- substituted Ci_₄ alkyl, amino-substituted Ci_ alkyl, methyl, trifluoromethyl, or COO(Ci_ alkyl);is optionally substituted with up to three groups selected from halo, Ci_ alkyl, Ci_ alkoxy, oxo, or -COO(d-4 alkyl);

R² is H or d_₄ alkyl;

T is (CH₂)₁.₃;

Y is selected from d-2 aminoalkyl, C .₆ heterocyclyl, and C₃.₆ cycloalkyl, wherein C1-2 aminoalkyl, C₄_e heterocyclyl, and d-e cycloalkyl are each optionally substituted with up to two groups selected from amino, oxo, halo, hydroxyl, Ci_ alkoxy, hydroxyl- substituted d-4 alkyl, amino-substituted d-4 alkyl, COOH, COO-(d-4 alkyl), and d_₃ haloalkyl;

A is NH, N(d_ alkyl), or a bond between the carbonyl in Formula (I I I) and Q;

Q is selected from d-4 alkyl, -(CH₂)₀-2-d-6heterocyclyl, -(CH₂)₀-2-d-6heteroaryl, and -(CH₂)o-2-phenyl, and Q is optionally substituted with up to three groups selected from halo, hydroxyl, amino, -SH, -R, -OR, -SR, -S0₂R, -NHR, and -NR₂, where each R is Ci_-6 alkyl optionally substituted with halo, -SH, -NH₂, OMe, or -OH.

Additional embodiments include compounds of Formula (III) wherein or R¹ is C₃.₅ alkyl substituted with -OH, -NH₂, -COOH, -COO(Ci-₄ alkyl), -CONMe₂, CONHMe, or - CONH₂; and all other features are as described above for Formula (III).

In these compounds, Z can be CH or N; in many embodiments, Z is CH.

In these compounds, Ar¹ can be a substituted phenyl as described above, typically a di-substituted phenyl such as dihalophenyl. In preferred embodiments, Ar¹ is a 2,5- dihalophenyl such as 2,5-difluorophenyl, 2-chloro-5-fluorophenyl, or 2-fluoro-5- chlorophenyl.

In these compounds, Ar² can be a substituted phenyl or pyridine as described above, or an optionally substituted cyclic either. In many embodiments, Ar² is an unsubstituted or mono-substituted phenyl or pyridine. Suitable substituents for the substituted Ar² include halo, hydroxyl, and amino; the substituent can be at any position, e.g., it can be at the position meta to the position of Ar² that is attached to the imidazole / triazole ring in the Formula.

In any of these embodiments of compounds of Formula (III), R² can be H or Ci_₄ alkyl, typically it is H or Me, preferably H.

In any of these embodiments of compounds of Formula (III), R¹ can be a substituted or unsubstituted heterocyclic group as described above; in some

embodiments, R¹ is a cyclic ether such as tetrahydropyran-4-yl, tetrahydropyran-3-yl, tetrahydrofuran-3-yl, or oxetan-3-yl. Tetrahydropyran-4-yl is sometimes preferred: when incorporated into an ADC, this moiety reduces aggregation of the conjugate that may occur when R¹ is a t-butyl, for example, so this moiety is especially advantageous for ADC purposes. Data demonstrating this advantage is included in Table 7 herein.

In any of these embodiments of compounds of Formula (III), T can be methylene, ethylene or propylene. In preferred embodiments, T is methylene when Y is one of the heterocyclic or cycloalkyl groups described, and T is methylene or -CH₂CH₂- when Y is an aminoalkyl group within the scope of Formula (III).

In any of these embodiments of compounds of Formula (III), A can be a bond; in other embodiments, A is preferably -NH-. In any of these embodiments of compounds of Formula (III), Y can be an aminoalkyi group or heterocyclic group as described above. In some embodiments, Y is an aminoalkyi such as 1 -fluoro-2-amino-2-ethyl or 1 -amino-2-ethyl or 1 -methoxy-2-amino- 2-ethyl. In some embodiments, Y is a pyrrolidine ring, e.g., pyrrolidin-3-yl, and may be substituted with F, CH₂F, CF₃, Me, or OH. In preferred embodiments, Y is a 3-pyrrolidinyl substituted at position 4 with one of these groups (F, CH₂F, CF₃, Me, or OH).

In any of these embodiments of compounds of Formula (III), R² can be H or Ci_₄ alkyl; in some embodiments R² is H or methyl, preferably H.

Some examples of Eg5 inhibitors for use in the immunoconjugates of the invention include any of the compounds in Table 1 , such as:

93

In certain embodiments of the compounds of Formula (II) or (III), R¹ is heterocyclic group such as a cyclic ether, e.g., a tetrahydropyranyl group (e.g. tetrahydropyran): a heterocyclic group at R¹ in the compounds of Formula (II) reduces aggregation when used as an ADC payload, as compared to conjugates having a t-butyl group as R¹, thus these compounds exhibit an advantage over known inhibitors of Eg5.

Linking Groups

The linking group L in Formula (I) can be a bond directly connecting payload compound X to Ab (i.e., L or each linker component can represent a bond connecting the groups flanking it together), or it can be a linking moiety comprising one or more linker components L₂, L₃, L₄, L₅, L₆, etc. Some preferred linking groups are depicted herein. Linking groups for ADCs commonly contain two or more linker components, which may be selected for convenience in assembly of the conjugate, or they may be selected to impact properties of the conjugate. Linker components include chemical groups that are readily formed when connecting Ab to X, such as thiol-maleimide groups, thioethers, amides, and esters; groups that are easily cleaved in vivo under conditions found in, on or around targeted cells, such as disulfides, hydrazones, dipeptides like Val-Cit, substituted benzyloxycarbonyl groups, and the like; spacers to orient X in a suitable position relative to Ab, such as phenyl, heteroaryl, cycloalkyl or heterocyclyl rings, and alkylene chains; and/or pharmacokinetic property-enhancing groups, such as alkylene substituted with one or more polar groups (carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide), and alkylene chains containing one or more -NH- or -O- in place of methylene group(s), such as glycol ethers (-CH₂CH₂0-)p where p is 1-10, which may enhance solubility or reduce intermolecular aggregation, for example.

A linking group may be divalent, meaning it can link only one X group to Ab, or it can be trivalent (able to link two X groups to Ab), or it can be polyvalent. Trivalent, tetravalent, and polyvalent linking group can be used to increase the loading of drug on an antibody, increasing the drug to antibody ratio (DAR) without requiring additional sites on the antibody for attaching linking groups. Such linking groups are known in the art, see e.g., Bioconjugate Chem., 1999 Mar-Apr; 10(2):279-88; US6638499; Clin Cancer Res October 15, 2004 10; 7063; WO2012/1 13847A1.

A linking group for use in the immunoconjugates of Formula (I) can be cleavable or non-cleavable. Cleavable linking groups such as those containing a hydrazone, a disulfide, the dipeptide Val-Cit, and ones containing a glucuronidase-cleavable p- aminobenzyloxycarbonyl moiety, are well known in the art, and can be used. See, e.g., Ducry, et al., Bioconjugate Chem., vol. 21 , 5-13 (2010). For these immunoconjugates, the linking group is substantially stable in vivo until the immunoconjugate binds to or enters a cell, at which point either intracellular enzymes or intracellular chemical conditions (pH, reduction capacity) cleave the linking group to free the Eg5 inhibitor.

Alternatively, non-cleavable linking groups can be used in the immunoconjugates of Formula (I). Non-cleavable linkers lack structural components designed to degrade in cells, and thus their structures can vary substantially. See, e.g., Ducry, et al.,

Bioconiuqate Chem., vol. 21 , 5-13 (2010). These immunoconjugates are believed to enter a targeted cell and undergo proteolytic degradation of the antibody rather than linking group decomposition; thus at least a portion of the linking group, and even some of the antibody or antibody fragment may remain attached to the Eg5 inhibitor. Formulas ( 11 A) and (MB) and (IIC) represent activated Eg5 inhibitors having a linking group attached at specific positions where it has been shown that residual parts of the linking group and/or antibody do not prevent inhibition of Eg5; thus attachment of a linking group at the positions represented by W in Formulas ( 11 A) and (MB) and (IIC) is preferred when a non- cleavable linking group is used.

Particularly suitable linking groups are those that reduce aggregation. Linking groups in Compounds 367 and 368 herein have been shown to have the effect of reducing aggregation when used with the Eg5 inhibitors described herein. Thus linking groups of the formula (V):

wherein R is of the formula -(CH₂)i-4-R , where R is a polar group selected from -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k-OCH₂CH₂OR²³, and - S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci_₄ alkyl; and j is an integer selected from 1 , 2, 3, and 4; and immunoconjugates Ab-L^*-X, comprising a payload (X) linked to an antibody (Ab), wherein the linking group L^* comprises a group of the formula -C(0)NR²¹- or -NR²¹-C(0)- , are also included in the invention. Preferably the linking group is of Formula (V). In Formula (V), [PL] indicates the attachment point of the payload, and [Ab] indicates the point of attachment to an antibody. The antibody is typically connected to L^* via a sulfur atom of a cysteine residue, which may be a cysteine from the native antibody sequence or a cysteine introduced by protein engineering. Preferred polar groups for these linkers include hydroxy and carboxy, j is typically 2, 3 or 4, and a R²¹ is often -(CH₂)₂-₃-R²³.

Table 1. Inhibitors of Eg5.

120



Compounds of Formula (IIA) and (MB) and (IIC)

The compounds of Formula (IIA) and (MB) and (IIC) comprise an Eg5 inhibitor attached to a reactive group and optionally one or more linker components connecting the Eg5 inhibitor to the reactive group. Table 2 depicts examples of these compounds, comprising an Eg5 inhibitor such as those shown in Table 1 plus a reactive functional group, and optionally one or more linker components.

Table 2. Payload-linking group Combinations before conjugation to Ab.

Cmpd # Payload + Linker Components + Reactive Functional Group

ı32

141

ı42

ı43







151

ı52

ı55





ı62

ı65



ı73

ı74

ı75

Cmpd # Payload + Linker Components + Reactive Functional Group

368

368

ı95

ı96

201

204

205

211

487

488

489

215

493

494

495

496

218

219

Entries 508 and 509 are provided as comparative examples, with Eg5 inhibitors known in the art but not within the scope of Formula II.

Antigen-Binding Moieties

The antigen-binding moiety in Formula (I) or (IA) can be any moiety that selectively binds to a cell-surface marker found on a targeted cell type. In some aspects, Ab is an antibody or antibody fragment (e.g., antigen binding fragment of an antibody) that specifically binds to an antigen predominantly or preferentially found on the surface of cancer cells, e.g., a tumor-associated antigen. In some aspects, Ab is an antibody or antibody fragment (e.g., antigen binding fragment) that specifically binds to a cell surface receptor protein or other cell surface molecules, a cell survival regulatory factor, a cell proliferation regulatory factor, a molecules associated with (for e.g., known or suspected to contribute functionally to) tissue development or differentiation, a lymphokine, a cytokine, a molecule involved in cell cycle regulation, a molecule involved in

vasculogenesis or a molecule associated with (for e.g., known or suspected to contribute functionally to) angiogenesis. A tumor-associated antigen may be a cluster differentiation factor (i.e., a CD protein). In some aspects of the invention, the antigen binding moiety of the invention specifically binds to one antigen. In some aspects of the invention, the antigen binding moiety of the invention specifically binds to two or more antigens described herein, for example, the antigen binding moiety of the invention is a bispecific or multispecific antibody or antigen binding fragment thereof.

Exemplary antibodies or antigen binding fragments include but are not limited to anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2 antibody, anti-cKit antibody, anti-EGFR antibody, anti-cathepsin D antibody, andti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti- CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti- Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD1- antibody, anti-CD1 1 c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA CD45 antibody, anti-CD45RO antibody, anti- CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chains antibody, anti- melanosomes antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody, and anti-Tn-antigen antibody.

In one embodiment, the antigen binding moiety of the antibody-drug conjugates (ADC) of Formula (I) or (IA) specifically binds to a receptor encoded by an ErbB gene. The antigen binding moiety may bind specifically to an ErbB receptor selected from EGFR, HER2, HER3 and HER4. The antigen binding moiety may be an antibody that will specifically bind to the extracellular domain (ECD) of the HER2 receptor and inhibit the growth of tumor cells which overexpress HER2 receptor. The antibody may be a monoclonal antibody, e.g. a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. A humanized antibody may be huMAb4D5-1 , huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab). The antibody may be an antibody fragment, e.g. a Fab fragment.

The antibody used in the examples herein has the heavy chain and light chain sequences listed in Table 3. The sequences are the same as those for trastuzumab, and the antibody is referred to herein as "trastuzumab" or "TBS". Trastuzumab is thus one suitable antibody for use in the immunoconjugates of Formula (I) or (IA). Table 3. Sequence for antibody TBS used in the following Examples.

Heavy EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR Chain IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1 )

Light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA Chain SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO:2)

Antigen-binding moieties in Formula I or IA include, but are not limited to, antibodies or antibody fragments (e.g., antigen binding fragments) against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify

transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.

Antibodies and antibody fragments (e.g., antigen binding fragment) useful for the immunoconjugates of the invention include modified or engineered antibodies, such as an antibody modified to introduce a cysteine residue or lysine residue in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or fragment for conjugation to an Eg5 inhibitor. Similarly, the antibodies or antibody fragments can be modified to incorporate Pel or pyrrolysine (Noren et al., (1989) Science 14;244(4901 ): 182-188; Mendel et al., (1995) Annu Rev Biophvs Biomol Struct. 24:435- 462) as sites for conjugation to an Eg5 inhibitor. Methods for conjugating such antibodies with payloads or linker-payload combinations are known in the art.

Antigen-binding moieties (e.g., antibodies and antigen binding fragments) useful in the invention may also have other modifications or be conjugated to other moieties, such as but not limited to polyethylene glycol tags, albumin, and other fusion polypeptide.

Production of the Antibody

The antibodies and antibody fragments (e.g., antigen binding fragments) of the invention can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies can be obtained by, e.g., hybridoma or recombinant production. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

The invention further provides polynucleotides encoding the antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementary determining regions as described herein.

The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below) encoding an antibody or its binding fragment. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981 ; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Eriich (Ed.), Freeman Press, NY, NY, 1992; PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991 ; and Eckert et al., PCR Methods and Applications 1 :17, 1991. Also provided in the invention are expression vectors and host cells for producing the antibodies or antibody fragments described above. Various expression vectors can be employed to express the polynucleotides encoding the antibody chains or binding fragments of the invention. Both viral-based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). For example, nonviral vectors useful for expression of the polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adenoassociated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding an antibody chain or fragment of the invention. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ,

metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an antibody chain or fragment of the invention. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al.. Meth. Enzvmol.. 153:516. 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted antibody sequences. More often, the inserted antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the antibody chains of the invention can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters may be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express the antibodies or antibody fragments of the invention. Insect cells in combination with baculovirus vectors can also be used.

In one aspect, mammalian host cells are used to express and produce the antibodies and antibody fragments of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes (e.g., the myeloma hybridoma clones as described in the Examples) or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y. , N.Y. , 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and

transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP poll 11 promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium

phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.

Immunoconiuqates

The invention provides immunoconjugates comprising an Eg5 inhibitor linked to an antigen-binding moiety, such as an antibody or antibody fragment. Preferred

immunoconjugates of the invention are those of Formula (I) or (IA) as described herein.

Methods for making such immunoconjugates are well known in the art. Preferred immunoconjugates include those disclosed in Tables 5 and 6, and variations thereof having another antigen binding moiety instead of trastuzumab, particularly such conjugates where trastuzumab is replaced by an antibody selected from the following list: anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2 antibody, anti-cKit antibody, anti-EGFR antibody, anti-cathepsin D antibody, andti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti- CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti- Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD1- antibody, anti-CD1 1 c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA CD45 antibody, anti-CD45RO antibody, anti- CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chains antibody, anti- melanosomes antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody, and anti-Tn-antigen antibody.

In some embodiments, an immunoconjugate of the invention comprises an antibody or antibody fragment Ab having antigen-binding activity, where the linking group L is attached to Ab at a cysteine sulfur atom of Ab:

where L and X are as defined for Formula (I), and R' and R" are side chains of amino acids adjacent to a cysteine in Ab. In these embodiments, -S-L- often comprises a thiol- maleimide linkage, and L optionally comprises additional linker components. In other embodiments, the conjugate is linked to X through a linking group comprising -S-CH₂- C(=0)-NH-L²-L³-L⁴-L⁵-L⁶-, where linker components L², L³, L⁴, L⁵, and L⁶ are as defined for Formula (IA). Methods for forming these conjugates by reaction of a compound having an alpha-halo acetamide or maleimide with the sulfur atom of a cysteine residue in the antigen binding moiety (antibody) are well known in the art.

Preferred immunoconjugates include immunoconjugates comprising any of the payload compounds in the following tables (Tables 5 and 6) conjugated with an antibody (AntiB), where the conjugate has the structure shown in the Table, wherein AntiB— S- represents an antibody bonded to the maleimide ring via a sulfur atom (S in the structure) of a cysteine residue of the antibody. In preferred embodiments, the antibody (AntiB) is an antibody that recognizes an antigen expressed on a cancer cell. Suitable antigens are disclosed herein, including anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-HER-2 antibody, anti-cKit antibody, anti-EGFR antibody, anti-cathepsin D antibody, andti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P- glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD1- antibody, anti-CD1 1 c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD39 antibody, anti-CD41 antibody, anti-LCA CD45 antibody, anti- CD45RO antibody, anti-CD45RA antibody, anti-CD71 antibody, anti-CD95/Fas antibody, anti-CD99 antibody, anti-CD100 antibody, anti-S-100 antibody, anti-CD106 antibody, anti- ubiquitin antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-lambda light chains antibody, anti-melanosomes antibody, anti-prostate specific antigen antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratins antibody, and anti-Tn-antigen antibody

The cysteine residue connecting the antibody to the maleimide compound may be naturally present in the native antibody, or it may have been introduced into the antibody by protein engineering methods known in the art. Antibodies engineered to contain a cysteine residue introduced by protein engineering are sometimes preferred. In particular, antibodies engineered to introduce cysteine in place of at least one of the following sites are particularly suited for use in the immunoconjugates of the invention: heavy chain sites K360, E152, and S375; and Light chain residue K107. In particular, the

combinations HC-K360C with LC-K107C, and HC-E152C with HC-S375C are well suited. (EU Numbering)

It is understood that the antibody may contain more than one payload: in typical embodiments, the conjugate contains 2-6, preferably 3-5 payload compounds (Eg5 inhibitors) on an antibody that consists of two heavy chain and two light chain peptides.

Table 5. Payload/Linker Combinations and Conjugates. AntiB is an antibody in preferred conjugates. Alternatively, AntiB represents a cysteine residue attached via its sulfur atom to the succinimide ring in the Conjugates.

232 Table 6. Additional Payload-Linker Compounds and Conjugates. AntiB is an antibody in preferred immunoconjugates. Alternatively, AntiB represents a cysteine residue attached via its sulfur atom to the succinimide ring in these Conjugates.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, and the like. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.

The immunoconjugates of the invention are typically formulated as solutions or suspensions in aqueous buffer and/or isotonic aqueous solution. They are typically formulated and administered at a pH near neutral to protect the stability of the protein component, e.g. at pH between 6 and 8, and may include pharmaceutically acceptable salts. And/or buffers Because the protein components are typically produced from cells, they may contain counterions found in cells, e.g., phosphate, acetate, sodium, potassium, and the like, and such counterions if present are typically not specifically identified or characterized. Moreover, they are typically isolated and handled in buffered solutions such as phosphate-buffered saline, and any counterions present are not expected to affect activity, The immunoconjugates are typically administered parenterally, either by injection or by infusion. Methods for their formulation and administration are similar to those for formulation and administration of other biologic-based pharmaceuticals such as antibody therapeutics, and are known to those of skill in the art.

Compounds of Formula (III) for use as small-molecule pharmaceuticals may be formulated for and administered by conventional routes, such as orally, topically, parenterally, buccally, by inhalation, or as suppositories.

Suitable compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide

pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient.

Suitable compositions for transdermal application include an effective amount of a compound of the invention with a suitable carrier. Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Suitable compositions for topical application, e.g., to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will in particular be appropriate for dermal application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sun creams, lotions, sprays and the like. They are thus particularly suited for use in topical, including cosmetic, formulations well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

As used herein a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, atomizer or nebulizer, with or without the use of a suitable propellant.

The present invention further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present invention as active ingredients, since water may facilitate the degradation of certain compounds.

Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous

compositions are packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e. g., vials), blister packs, and strip packs.

The invention further provides pharmaceutical compositions and dosage forms that comprise one or more agents that reduce the rate by which the compound of the present invention as an active ingredient will decompose. Such agents, which are referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers, etc.

The compounds (immunoconjugates) of formula I in free form or in salt form, exhibit valuable pharmacological activities: as the data herein demonstrates, the compounds of Formula (II) and (III) inhibit growth of tumor cells, and are accordingly useful to treat cancers. As the data further demonstrate, these compounds can advantageously be delivered as the payload of an ADC. Such conjugates, as demonstrated herein, exhibit substantial activity on targeted cells in vitro and on tumors in vivo, as demonstrated by potent growth inhibition of xenograft tumors representing different human cancers. Thus the immunoconjugates of the invention, comprising a payload of Formula (II) or (III) linked to an antigen binding moiety such as an antibody, are also useful to treat cancers, such as glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, renal, gastric (e.g., gastrointestinal stromal tumors (GIST)), liver, and pancreatic cancer, and sarcoma.

The compounds and immunoconjugates of the invention are particularly useful for treating cancers known in the art to be inhibited by compounds active against Eg5, and those tumor types demonstrated herein to be susceptible to inhibition by the compounds and conjugates of the invention. Suitable indications for treatment include, but are not limited to, gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, renal, gastric (e.g., gastrointestinal stromal tumors (GIST)), liver, and pancreatic cancer, and sarcoma.

Thus, as a further embodiment, the invention provides the use of a compound of formula (I) or (III) or any of the embodiments within the scope of Formula (I) and (III) as described herein, in therapy. In a further embodiment, the therapy is for a disease which may be treated by inhibition of Eg5. In another embodiment, the compounds of the invention are useful to treat cancers, including but not limited to breast cancer, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, myelogenous leukemia, lymphocytic leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), hairy cell leukemia and multiple myeloma.

The methods typically comprise administering an effective amount of a compound as described herein or a pharmaceutical composition comprising such compound to a subject in need of such treatment. The compound may be administered by any suitable method such as those described herein, and the administration may be repeated at intervals selected by a treating physician.

Thus, as a further embodiment, the present invention provides the use of a compound of formula (I) or (III), or any of the embodiments of such compounds described herein, for the manufacture of a medicament. In a further embodiment, the medicament is for treatment of a disease which may be treated by inhibition of Eg5. In another embodiment, the disease is selected from gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, melanoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (e.g., chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL), lymphoma (especially non-Hodgkin's), bladder, renal, gastric (e.g., gastrointestinal stromal tumors (GIST)), liver, and pancreatic cancer, and sarcoma.

The pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

A pharmaceutical combination refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present invention and a combination partner (e.g. another drug as explained below, also referred to as

"therapeutic agent" or "co-agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of the present invention and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of the present invention and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides

therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

The above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof. The compounds of the present invention can be applied in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either enterally, parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution. The dosage in vitro may range between about 10 ³ molar and 10^"9 molar concentrations. A therapeutically effective amount in vivo may range depending on the route of administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg. The compound of the present invention may be administered either

simultaneously with, or before or after, one or more therapeutic co-agent(s). The compound of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the co-agent(s).

In one embodiment, the invention provides a product comprising a compound of formula (I) and at least one other therapeutic co-agent as a combined preparation for simultaneous, separate or sequential use in therapy. In one embodiment, the therapy is the treatment of a disease or condition mediated by Eg5, such as cancer. Products provided as a combined preparation include a composition comprising the compound of formula (I) or (III) and the other therapeutic co-agent(s) together in the same

pharmaceutical composition, or the compound of formula (I) or (III) and the other therapeutic co-agent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical composition comprising a compound of formula (I) or (III) and another therapeutic co-agent(s).

Optionally, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, as described above.

Suitable co-agents for use with the compounds and conjugates of the invention include other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics), pain relievers, anti-inflammatory agents, cytoprotective agents, and combinations thereof.

Specific co-agents considered for use in combination with the compounds and conjugates disclosed herein include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin

(Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-

Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride

(Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide

(Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea

(Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L- asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

Some patients may experience allergic reactions to the compounds of the present invention and/or other anti-cancer agent(s) during or after administration; therefore, antiallergic agents are often administered to minimize the risk of an allergic reaction. Suitable anti-allergic agents include corticosteroids, such as dexamethasone (e.g., Decadron®), beclomethasone (e.g., Beclovent®), hydrocortisone (also known as cortisone, hydrocortisone sodium succinate, hydrocortisone sodium phosphate, and sold under the tradenames Ala-Cort®, hydrocortisone phosphate, Solu-Cortef®, Hydrocort Acetate® and Lanacort®), prednisolone (sold under the tradenames Delta-Cortel®, Orapred®, Pediapred® and Prelone®), prednisone (sold under the tradenames Deltasone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone (also known as 6- methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, sold under the tradenames Duralone®, Medralone®, Medrol®, M-Prednisol® and Solu- Medrol®); antihistamines, such as diphenhydramine (e.g., Benadryl®), hydroxyzine, and cyproheptadine; and bronchodilators, such as the beta-adrenergic receptor agonists, albuterol (e.g., Proventil®), and terbutaline (Brethine®).

Some patients may experience nausea during and after administration of the compound of the present invention and/or other anti-cancer agent(s); therefore, antiemetics are used in preventing nausea (upper stomach) and vomiting. Suitable antiemetics include aprepitant (Emend®), ondansetron (Zofran®), granisetron HCI (Kytril®), lorazepam (Ativan®, dexamethasone (Decadron®), prochlorperazine (Compazine®), casopitant (Rezonic® and Zunrisa®), and combinations thereof.

Medication to alleviate the pain experienced during the treatment period is often prescribed to make the patient more comfortable. Common over-the-counter analgesics, such Tylenol®, are often used. However, opioid analgesic drugs such as hydrocodone/paracetamol or hydrocodone/acetaminophen (e.g., Vicodin®), morphine (e.g., Astramorph® or Avinza®), oxycodone (e.g., OxyContin® or Percocet®), oxymorphone hydrochloride (Opana®), and fentanyl (e.g., Duragesic®) are also useful for moderate or severe pain. In an effort to protect normal cells from treatment toxicity and to limit organ toxicities, cytoprotective agents (such as neuroprotectants, free-radical scavengers, cardio protectors, anthracycline extravasation neutralizers, nutrients and the like) may be used as an adjunct therapy. Suitable cytoprotective agents include Amifostine (Ethyol®), glutamine, dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® or Totect®), xaliproden (Xaprila®), and leucovorin (also known as calcium leucovorin, citrovorum factor and folinic acid).

In one embodiment, the invention provides a kit comprising two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I) or (III). In one embodiment, the kit comprises means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is a blister pack, as typically used for the packaging of tablets, capsules and the like.

The kit of the invention may be used for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit of the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of the invention and the other therapeutic co-agent may be manufactured and/or formulated by the same or different manufacturers. Moreover, the compound of the invention and the other therapeutic may be brought together into a combination therapy: (i) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the invention and the other therapeutic agent); (ii) by the physician themselves (or under the guidance of the physician) shortly before administration; (iii) in the patient themselves, e.g. during sequential administration of the compound of the invention and the other therapeutic agent.

Accordingly, the invention provides the use of a compound of formula (I) or (III) for treating a disease or condition mediated by Eg5, wherein the medicament is prepared for administration with another therapeutic agent. The invention also provides the use of another therapeutic co-agent for treating a disease or condition, wherein the medicament is administered with a compound of formula (I) or (III).

The invention also provides a compound of formula (I) or (III) for use in a method of treating a disease or condition mediated by Eg5, wherein the compound of formula (I) or (III) is prepared for administration with another therapeutic agent. The invention also provides another therapeutic co-agent for use in a method of treating a disease or condition mediated by Eg5, wherein the other therapeutic co-agent is prepared for administration with a compound of formula (I) or (III). The invention also provides a compound of formula (I) or (III) for use in a method of treating a disease or condition mediated by Eg5, wherein the compound of formula (I) or (III) is administered with another therapeutic co-agent. The invention also provides another therapeutic co-agent for use in a method of treating a disease or condition mediated by Eg5, wherein the other therapeutic co-agent is administered with a compound of formula (I) or (III).

The invention also provides the use of a compound of formula (I) or (III) for treating a disease or condition mediated by Eg5, wherein the patient has previously (e.g. within 24 hours) been treated with another therapeutic agent. The invention also provides the use of another therapeutic agent for treating a disease or condition mediated by Eg5, wherein the patient has previously (e.g. within 24 hours) been treated with a compound of formula (I) or (III).

Synthetic Methods

All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesize the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art (see e.g., Houben-Weyl 4th Ed. 1952, Methods of Organic Synthesis, Thieme, Volume 21 ). Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art in view of the following examples.

Many Eg5 inhibitor compounds of formula (II) can be prepared according to methods known in the art, including methods disclosed in WO2007/021794,

WO2006/002236, WO2008/063912, WO2009/077448, WO201 1/128381 , and

WO201 1/128388.

Compounds of Formula (III) can be prepared similarly, using known methods in combination with methods described herein. Illustrative examples of synthesis of these compounds are provided in the following general Schemes. Scheme 1.

This process begins with the known protected chiral amino acid having a THP group at the alpha-carbon, and formation of an ester with an appropriate alpha-halo acetophenone to provide the desired Ar¹ group. Treatment with ammonium acetate provides the substituted imidazole with retention of chirality at the group on C-2 of the imidazole ring. The imidazole nitrogen can be alkylated with mild base to introduce

Ar²CH₂-. Deprotection of the carbamate provides a free amine, which can be alkylated with a suitable primary alcohol, by oxidation with Dess-Martin periodinane (DMP) or similar oxidation and Schiff base formation, followed by reduction of the imine using cyanoborohydride or a similar reducing agent. In the Scheme, this step introduces a pyrrolidine ring, but other groups within the scope of Formula (II) or (III) can be introduced similarly to provide other compounds with different -T-Y groups for the compounds of Formula (I I) or (I I I). Protecting groups may be utilized as needed to allow various R¹⁰ groups to be present on the pyrrolidine ring, for example, or to accommodate

substitutions on Ar¹ or Ar² as needed.

The free amine can then be acylated with any suitable acylating agent to introduce appropriate -A-Q moieties using conventional methods known in the art. In the illustrative example in the Scheme, a chiral lactate derivative is used to introduce an acyl group in protected form, having a bond for the group A in Formula (I I) and (I I I), and a protected hydroxyalkyl group for group Q. Deprotection of the pyrrolidine ring nitrogen and of the hydroxyl group on Q provides a compound of Formula (I I I).

Many suitable pyrrolidine rings for making compounds of the invention are known in the art, and Scheme 2 depicts a synthesis for certain of these. The known 3-pyrroline ring is protected with a carbobenzyloxy (CBZ) or other suitable protecting group, and oxidized with meta-chloro peroxybenzoic acid. The epoxide is then opened with a

Grignard reagent, such as vinyl Grignard, in the presence of copper (I) bromide to provide a trans-disubstituted pyrrolidine. This can be used to make various pyrrolidine

intermediates, and its enantiomers can be readily separated. It can be used, for example, as depicted in Scheme 2, to prepare a fluorinated pyrrolidine ring. The hydroxyl group can be replaced by F, with inversion of stereochemistry, using a fluorination reagent such as perfluoro-1 -butanesulfonyl fluoride (PBSF) or DAST. The vinyl group can then be oxidized with osmium tetroxide, and the resultant aldehyde can be reduced by

conventional methods such as sodium borohydride to provide the chiral pyrrolidine used in Scheme 1 .

Scheme 2. Synthesis of pyrrolidine intermediates.

^MgBr /Pr₂EtN, PSBF, HF.Et₃N

PhCF₃, 81 %

Scheme 3.

Scheme 3 illustrates synthesis of compounds wherein A is -NH-, beginning with an intermediate from Scheme 1. Phosgene in dichloromethane followed by introduction of a suitable amine provides the protected intermediate, and deprotection can be

accomplished under conventional conditions, e.g., palladium on carbon in methanol, using ammonium formate or hydrogen. Scheme 3A.

1 ) i) bis(para-nitrophenol) carbonate

Scheme 3A illustrates a method to synthesize compounds of Formula (IIA), having a reactive functional group (maleimide, in this case) attached through the acyl group of the compound of Formula (II), i.e., L is attached to Q. The intermediate shown, from Scheme 2, is converted to an activated acylating agent using bis-(para-nitrophenol)carbonate, forming a mixed carbonate with a p-nitrophenoxy leaving group. The mixed carbonate is then allowed to react with a suitable amine, followed by deprotection of the pyrrolidine ring nitrogen to provide a compound of Formula (IIA), wherein W is a maleimide, suitable for reaction with a thiol on Ab, or on a linker component attached to Ab. The product in this example would be considered a non-cleavable linker, since none of the linker components present are designed for in vivo cleavage at a rate faster than the rate of degradation of an antibody to which the moiety would be attached in an ADC of Formula (I).

Scheme 3B.

Scheme 3B illustrates a method to prepare compounds of Formula (MB), using the product of Scheme 1. In this method, a mixed carbonate of the maleimide-containing linking group precursor is formed, using bis(para-nitrophenol)carbonate. The mixed carbonate is then used to acylate the pyrrolidine nitrogen of the Eg5 inhibitor, providing the compound of Formula (MB) shown above. In this example, the reactive functional group of W in Formula (MB) is a maleimide, and the linker components in group W include a cleavable linker (val-cit), so this compound exemplifies a conjugate having a cleavable linking group that is subject to cleavage by a cathepsin B. The para- aminobenzyloxycarbamate linker component functions as a self-immolative linker: once cathepsin B cleaves the val-cit dipeptide from the para-amino group, the benzyl carbamate spontaneously decomposes to release the Eg5 compound.

The invention further includes any variant of the present processes, in which an intermediate product obtainable at any stage thereof is used as starting material and the remaining steps are carried out, or in which the starting materials are formed in situ under the reaction conditions, or in which the reaction components are used in the form of their salts or optically pure material.

The following examples are intended to illustrate the invention and are not to be construed as being limitations thereon. Temperatures are given in degrees Celsius. If not mentioned otherwise, all evaporations are performed under reduced pressure, typically between about 15 mm Hg and 100 mm Hg (= 20-133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, NMR.

Abbreviations used are those conventional in the art.

DCM: dichloromethane

DIAD: Diisopropyl azodicarboxylate

DIPEA: diisopropyl ethylamine

DMF: N,N-dimethylformamide

ETOAC: ethyl acetate

TBSCI: tert-butyldimethylsilyl chloride

TFA: trifluoroacetic acid

THF: tetrahydrofuran

All reactions were carried out under Ar using commercial grade solvents without any further distillation. Reagents were used as commercial grade without further purification. Thin layer chromatography was carried out using TLC-aluminum sheets with 0.2 mm of silica gel (Merck F254). Column chromatography was carried out using an ISCO Combiflash Companion system, using flash grade prepacked Redisep® columns.

NMR spectra were recorded at 23°C or 29°C using the following spectrometers: Bruker 400 MHz and Bruker AVANCE 600 MHz proton frequency, equipped with a 1.7 mm ¹H{¹³C,¹⁵N} CryoProbe™. Preparative HPLC was performed on Waters

Autopurification system using the following conditions: Column Sunfire C18 30 x 100mm, 5μ, gradient elution with CH₃CN in water + 0.1 %TFA-CH₃CN at 30 ml/min.

LC/MS data were produced with a Waters Acquity UPLC/SQD system, using a photodiode array detector and a single quadrupole mass detector. The following conditions were utilized:

Column: Waters Acquity HSS T3 1.8 μιη 2.1 x 50 mm

Eluent A: Water + 0.05% Formic acid + 3.75mM Ammonium acetate

Eluent B: Acetonitrile +0.04% Formic Acid

Column temperature: 60°C

Injection-Vol. 1 μΙ, partial loop

PDA Full scan 210 - 450 nm and one user-selectable wavelength

Method A: LCMS_2_MINUTES

Flow 1.0 ml/min

Stop Time 2.00 min

Gradient: Time % A (Eluent A) % B (Eluent B)

0.00 95 5

1.40 2 98

1.80 2 98

1.90 95 5

2.00 95 5

Mass range ESI +/-: 100 - 1200 m/z

Method B: LCMS_10_MINUTES

Flow 1.0 ml/min

Stop Time 10.00 min

Gradient: Time % A (Eluent A) % B (Eluent B)

0.00 95 5

9.40 2 98 9.80 2 98

9.90 95 5

10.00 95 5

Mass range ESI +/-: 100 - 1600 m/z

Method C: LC-MS Column Acquity UPLC BEH C18 1.7um, 2.1^*50mm

5 minutes (Flow 0.7 ml/min, solvent A: water+0.1 % formic acid, solvent B: ACN+0.1 % formic acid, Gradient: from 20 to 100% B in 4.3 min)

Where the LC method is not specified, Method A was used if retention time is under 1.5 minutes and Method B was used for retention times between 1.5 and 10 minutes.

UPLCMS - Method D (Polar Method, 2 mins Run):

System: Waters Acquity UPLC with Waters SQ detector.

Column: Acquity HSS T3 1.8 μιη 2.1 x 50 mm.

Flow: 1 ml/min. Column temperature: 60 °C.

Gradient: from 1 to 98% B in 1.4 min, A = water + 0.05 % formic acid + 3.75 mM ammonium acetate, B = acetonitrile + 0.0.4 % formic acid.

UPLCMS Method E (4 minute runs)

System: Waters Acquity UPLC with Waters SQ detector.

Column: Sunfire C18 3.5 μιη 2.1x20mm.

Flow: 0.62 ml/min. Column temperature: 40 °C.

Gradient: from 5 to 100% B in 4 min, A = water + 0.1 % trifluoroacetic acid, B = acetonitrile + 0.1 % trifluoroacetic acid.

Preparative LC Methods

Normal Phase Chromatography - PrepLC Method A

System: CombiFlash Rf200. Column:RediSep Column Silica.

Gradiend: fromO to 100% B, A = Heptane, B = Ethyl Acetate. Normal Phase Chromatography - PrepLC Method B System: CombiFlash Rf200. Column:RediSep Column Silica.

Gradiend: fromO to 100% B, A = Dichloromethane, B = MeOH. Preparative Reverse Phase Chromatography - PrepLC Method C System: Waters Acquity Prep LC/MS with Waters SQ detector. Column: Sunfire Preparative C18, 5 μιη, 30 x 100 mm. Flow: 30 ml/min.

Gradient: from 5 to 100% B, A = acetonitrile + 0.1 % trifluoroacetic acid, B + water + 0.1 % trifluoroacetic acid.

Preparative Reverse Phase Chromatography - PrepLC Method D System: Waters Acquity Prep LC/MS with Waters SQ detector. Column: Sunfire Preparative C18, 5 μιη, 30 x 150 mm. Flow: 60 ml/min.

Synthesis of Selected Intermediates

Benzyl 2,5-dihvdro-1 H-pyrrole-1-carboxylate

To a solution of 2,5-dihydro-1 H-pyrrole (30 g, 434 mmol, 96% from Alfa Aesar) in dioxane (1000 ml_, 0.43 M solution) was added CbzOSu (130 g, 521 mmol). After being stirred at room temperature for 18 h, the reaction mixture was concentrated to around 300 ml_, diluted with 1000 ml_ of EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na₂S0₄, filtered, and concentrated in vacuo. The desired benzyl 2,5- dihydro-1 H-pyrrole-1-carboxylate was obtained in 91 % yield (80.0 g) as a colorless oil by flash column chromatography. Rf = 0.6 (30% EtOAc in hexanes). ¹H NMR (CDCI_3, 400 MHz): 57.32 (5H, m), 5.80 (2H, m), 5.77 (2H, s), 4.22 (4H, m). LC/MS (uplc): MhT 204.2, 160.1 (-44), 0.86 min.

Benzyl 6-oxa-3-azabicvclo[3.1.Olhexane-3-carboxylate

z

To a solution of benzyl 2,5-dihydro-1 H-pyrrole-1-carboxylate (33 g, 163 mmol; 90% from Aldrich) in dichloromethane (540 ml_, 0.3 M solution) was added m-CPBA (44 g, 340 mmol, 77% from Aldrich). After the reaction mixture was stirred at room temperature for 18 h, 500 ml_ of saturated Na₂C0₃ aqueous solution was added and the resulting mixture was stirred at room temperature for 1 h. The organic layer was separated, washed with water and brine, dried over anhydrous Na₂S0₄, filtered, and concentrated in vacuo. The desired product as a yellow oil was obtained in 83% yield (29.5 g) by flash column chromatography. Rf = 0.5 (30% EtOAc in hexanes). ¹H NMR (CDCI₃, 400 MHz): δ 3.38 (2H, dd, J = 12.8, 6.0 Hz ), 3.68 (2H, d, J = 3.6 Hz), 3.87 (2H, dd, J = 13.2, 19.6), 5.1 1 (2H, s), 7.33( 5H, m). LC/MS (uplc): MH⁺ 220.0, 0.69 min.

Benzyl 3-hvdroxy-4-vinylpyrrolidine-1-carboxylate

To a solution of benzyl 6-oxa-3-azabicyclo[3.1.0]hexane-3-carboxylate (28.5 g, 130 mmol) and CuBr SMe₂ (26.7 g, 130 mmol) in anhydrous THF (260 ml_, 0.5 M solution) at - 40 °C was slowly added vinyl magnesium bromide (520 ml_, 1.0 M solution in THF). The reaction mixture was then warmed up to - 20 °C for 2 h. After quenched with saturated NH₄CI aqueous solution (200 ml_), the reaction mixture was extracted with EtOAc (500 ml_). The organic layer was washed with water and brine, dried over anhydrous Na₂S0₄, filtered and concentrated in vacuo. The desired racemic mixture of trans-(±)-benzyl 3- hydroxy-4-vinylpyrrolidine-1-carboxylate was obtained in 48% yield (15.5 g) as a yellow oil by flash column chromatography. Rf = 0.2 (30% EtOAc in hexanes). ¹H NMR (CDCI_3, 400 MHz): δ 2.71 (1 H, m), 3.28 (2H, m), 3.72 (2H, m), 4.1 1 (1 H, m), 5.14 (2H, s), 5.16-5.23 (2H, m), 5.69 (1 H, m), 7.33 (5H, m). LC/MS (uplc): MH⁺ 248.0, 0.78 min.

Resolution of trans-(±)-benzyl 3-hvdroxy-4-vinylpyrrolidine-1-carboxylate

racemic mixture desired

The racemic mixture of trans-(±)-benzyl 3-hydroxy-4-vinylpyrrolidine-1-carboxylate (14 g) was submitted to the Separation Laboratory in Basel (contact: Dr. Eric Francotte, Tel. +41 6169 62971 ). The desired enantiomerically enriched (3S,4R)-benzyl 3-hydroxy-4- vinylpyrrolidine-1-carboxylate (6.3 g, >99.5%ee) and undesired (3R,4S)-benzyl 3-hydroxy- 4-vinylpyrrolidine-1-carboxylate (6.7 g, 99.5 %ee) were obtained with 92% recovery.

(3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylate

bz

To a solution of (3S,4R)-benzyl 3-hydroxy-4-vinylpyrrolidine-1-carboxylate (5.0 g, 20.2 mmol) in PhCF₃ (81 mL, 0.25 M solution) was added N,N-diisopropylethylamine (53 mL, 303 mmol), triethylamine trihydrofluoride (19.8 mL, 121 mmol) and perfluoro-1- butanesulfonyl fluoride (PBSF, 3.6 mL, 20.2 mmol). The resulting mixture was stirred at room temperature. After 60 and 120 minutes, additional perfluoro-1-butanesulfonyl fluoride (3.6 mL, 20.2 mmol) was added. After 18 hours, the reaction mixture was transferred to a separatory funnel and was washed twice with 50 mL of 1.0 N HCI (Caution! lots of heat produced), twice with saturated NaHC0₃ aqueous solution, and once with H₂0 and brine. The organic phase was dried over anhydrous Na₂S0₄, filtered and concentrated to provide a crude brown oil. The pure (3R,4R)-benzyl 3-fluoro-4- vinylpyrrolidine-1-carboxylate was obtained in 81 % yield (4.1 g) as a yellow oil by flash column chromatography (Si0₂, 10%-30% EtOAc in hexanes). Rf = 0.55 (30% EtOAc in hexanes). ¹H NMR (CDCI_3, 400 MHz): δ 7.37-7.25 (5H, m), 5.9 (1 H, m), 5.24 (2H, m), 5.14 (2H, m), 5.03 (1 H, dt, J = 52.8, 3.2 Hz), 3.9-3.5 (3H, m), 3.53 (1 H, q, J = 10.4 Hz), 2.83 (1 H, m). ¹³C NMR (CDCI_3, 100 MHz): δ 154.7, 154.6, 136.6, 131.89, 131.83, 128.48, 128.02, 127.94, 1 19.00, 1 18.94, 95.23, 94.47, 93.42, 92.67, 66.99, 66.94, 53.16, 52.94, 52.83, 52.60, 48.17, 48.02, 47.91 , 47.83, 47.2, 47.1. LC/MS (uplc): MH⁺ 250.0, 0.93 min.

(3R,4S)-benzyl 3-fluoro-4-(hvdroxymethyl)pyrrolidine-1-carboxylate

To a solution of (3R,4R)-benzyl 3-fluoro-4-vinylpyrrolidine-1-carboxylate (1.78 g, 7.15 mmol) in CH₃OH and H₂0 (2:1 , 18 mL) was added a solution of Os0₄ in H₂0 (3 mL of a 4% w/v solution, 0.5 mmol). Nal0₄ (4.6 g, 21.5 mmol) was then added in a single portion and the resulting mixture was stirred at room temperature. After 2 hours, the mixture was filtered to remove precipitated white solids and the filter cake was washed with EtOAc. The filtrate was concentrated in vacuo to remove the majority of the organic solvents. The residue was extracted with three portions of EtOAc and the combined organic layer was dried over anhydrous Na2SC>4, filtered and concentrated. The crude (3R,4S)-benzyl 3-fluoro-4-formylpyrrolidine-1-carboxylate was used for the next step without further purification. LC/MS (uplc): MH⁺ 208.2 (-44), 0.69 min.

To an ice-cooled solution of the above the crude of (3R,4S)-benzyl 3-fluoro-4- formylpyrrolidine-1-carboxylate in CH₂CI₂ (20 ml_), was added NaBH₄ (330 mg, 14.30 mmol). The reaction was stirred at room temperature. Upon completion of the reaction, the crude mixture was acidified with 0.5 M HCI and stirred for 30 min. The reaction mixture was partitioned between CH₂CI₂ and water. The organic layer was washed with sat. NaHC0₃ (twice) and water (twice), then dried under Na₂S0₄ and solvent evaporated under reduce pressure to give the desire product (3R,4S)-benzyl 3-fluoro-4- (hydroxymethyl)pyrrolidine-l-carboxylate (1.7 g, 6.9 mmol, 97%) as an oil. The crude was used without any further purification in the next step. LC/MS (uplc): MH+ 254.2, 210.2 (- 44), 0.78 min.

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-2-(tetrahvdro-2H- pyran-4-yl)acetate.

To an ice-cooled solution of (R)-2-((tert-butoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4- yl)acetic acid (4.82 g. 18.6 mmol) and K₂C0₃ (2.3 g, 16.7 mmol) in Acetone (372 ml_), was added 2-chloro-1-(2,5-difluorophenyl)ethanone (4.25 g, 22.3 mmol) followed by Kl (0.77 g, 4.6 mmol). The reaction was allowed to reach room temperature while stirring. Upon completion of the reaction by LC/MS (uplc, Method A), the mixture was cooled down to 0° C, and quenched with cold water (600 mL). After stirring for 15 min at 0°C, the reaction mixture was filtered and the precipitate was washed with acetone/H₂0 (1/3) to provide (R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-2- (tetrahydro-2H-pyran-4-yl)acetate as a solid (4.1 g, 9. 71 mmol, 52%). The solid was dried under high vacuum overnight, and was used in the next step without any further purification.

¹H-NMR (CDCI3, 600 MHz): δ 7.66 (1 H, m), 7.61 (1 H, m), 7.49 (1 H, m), 7.32 (1 H, m), 5.33 (2H, m), 4.06 (1 H, m), 3.86 (2H, m), 3.24 (2H, m), 2.01 (1 H, m), 1.75-1.4 (13H, m). LC/MS (uplc): MH+ 414.1 , 1.1 1 min (Method A). (R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4- vDmethvDcarbamate.

To a solution of (R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-2- (tetrahydro-2H-pyran-4-yl)acetate (4.1 g, 9.71 mmol) in Toluene (50 mL) was added ammonium acetate (15 g, 194 mmol). The resulting solution was heated under reflux (1 10 °C). Upon completion of the reaction by LC/MS (uplc, Method A), the mixture was cooled down to room temperature and partioned between EtOAC and H₂0. The organic layer was separated and washed with H₂0 (twice) and sat. solution NaHC0₃ (twice), then dried over Na2S04, filtered, and evaporated under reduce pressure to give crude (R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)carbamate as a brown solid (4 g, 9.7 mmol. 99%). The solid was dried under high vacuum overnight, and was used in the next step without any further purification. LC/MS (uplc): MH+ 394.2, 0.99 min (Method A).

(R)-tert-butyl ((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4- vDmethvDcarbamate.

To an ice-cooled solution of (R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)carbamate (4g, 9.7 mmol) and K₂C0₃ (2.8 g, 20.4 mmol) in DMF (68 mL), was added benzyl bromide (1.4 mL, 1 1.2 mmol) and the reaction stirred at 0°C for 1 h, followed by stirring at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), 80 mL of water were added and upon addition a solid precipitates. The solid was filtered and washed with DMF/H₂0 (1/1 ) to provide (R)- tert-butyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)carbamate (3. 7 g, 7.6 mmol, 73%). The solid was dried under high vacuum overnight, and was used without any further purification in the next step. ¹H-NMR (CDCI3, 400 MHz): δ 7.85 (1 H, m), 7.36 (4H, m), 7.21 (2H, m), 7.05 (1 H, m), 6.87 (1 H, m), 5.30 (1 H, m), 5.22 (1 H, m), 5.13 (1 H, d, m), 4.69 (1 H, m), 4.00 (1 H, m), 3.81 (1 H, m), 3.30 (2H, m), 2.13 (1 H, m), 1.80 (1 H, m), 1.45 (9H, m), 1.30 (1 H, m), 1.01 (1 H, m). LC/MS (uplc): MH+ 484.3, 1.34 min (Method A).

(R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4- vPmethanamine.

To an ice-cooled solution of (R)-tert-butyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)carbamate (3.7 g, 7.6 mmol) in CH₂CI₂ (80 mL), was added trifluoroacetic acid (20 mL). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude was diluted in CH₂CI₂, and the mixture partioned between CH₂CI₂ and sat. solution NaHC0₃. The organic layer was separated, and washed with sat. solution NaHC0₃ (twice) and water (twice), then dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure to give crude (R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran- 4-yl)methanamine (2.8 g, 7.3 mmol, 95%) as TFA salt. The crude was dried under high vacuum overnight, and used without any further purification in the next step. LC/MS (uplc): MH+ 384.2, 0.83 min (Method A).

(3R.4R)-benzyl 3-((((R)-(1-benzyl-4-(2.5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H- Pyran-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate.

To a solution of (3R,4S)-benzyl 3-fluoro-4-(hydroxymethyl)pyrrolidine-1-carboxylate (2.9 g, 1 1.4 mL) in CH₂CI₂ (80 mL) was added Dess-Martin periodinane (6.5 g, 15.20 mmol). The reaction mixture was stirred at room temperature for 30 min. Upon completion of the reaction, the crude product (3R,4S)-benzyl 3-fluoro-4-formylpyrrolidine-1 -carboxylate was used as a solution in the next step without further treatment.

To a solution of (R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methanamine (2.9 g, 7.6 mmol), and sodium triacetoxyborohydride (8.1 g, 38 mmol) in CH₂CI₂ (60 mL) was added the solution of (3R,4S)-benzyl 3-fluoro-4- formylpyrrolidine-1-carboxylate in CH₂CI₂ from the previous step. The reaction mixture was stirred at room temperature for 2 h. Upon dilution of the reaction mixture in CH₂CI₂, it was partioned between CH₂CI₂ and H₂0. The organic layer was separated and washed with sat. solution NaHC0₃ (twice) and H₂0 (twice), then dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. The desire (3R,4R)-benzyl 3-((((R)-(1-benzyl- 4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)- 4-fluoropyrrolidine-1-carboxylate was obtained after purification by normal phase column chromatography (PrepLC Method A, 2.7 g, 4.5 mmol, 59%). LC/MS (uplc): MH+ 619.3, 1.23 min (Method A).

General Procedure for N-Acylation of Intermediates To an ice-cooled solution of the amine (1 mmol) in CH₂CI₂ (0.1 M), was added /Pr₂EtN (7 mmol), followed by the acyl chloride (6 mmol). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the mixture was diluted in CH₂Cl₂ and partitioned between CH₂CI₂ and H₂0. The organic layer was separated and washed with sat. solution NaHC0₃ (twice) and H₂0 (twice), then dried over Na₂S0₄, filtered, and solvent evaporated under reduced pressure. The desired Cbz- Amide Payload was obtained after purification by normal phase column chromatography (PrepLC Method A or C).

Synthesis of Selected Linkers

Linker 1.

: TBSC1, imidazole, DMF, room temperature, 53% yield; b: maleimide, DIAD, PPh

-78...RT, THF, toluene, 62% yield; c: CF₃COOH, CH₂C1₂, 100% yield.

tert-Butyl (2-((tert-butyldimethylsilyl)oxy)ethyl)(2-hydroxyethyl)carbamate

Prepared as described in Liang, Qiren; De Brabander, Jef K..Tetrahedron, 2011 , vol. 67, pp. 5046 - 5053. tert-Butyl (2-((tert-butyldimethylsilyl)oxy)ethyl)(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)ethyl)carbamate

Under N₂: to a stirred solution of DIAD (0.237 mL, 1.221 mmol) and triphenylphosphine (320 mg, 1.221 mmol) at -78°C in 10 mL of toluene a freshly prepared solution of maleimide (1 18 mg, 1.221 mmol) and tert-Butyl (2-((tert-butyldimethylsilyl)oxy)ethyl)(2- hydroxyethyl)carbamate (300 mg, 0.939 mmol) in 10 mL of THF was added. The mixture was allowed to warm to RT and stirred overnight, diluted with DCM, and washed with water. Organics were dried with Na₂S0₄, filtered and absorbed onto Isolute. The desired product was obtained after purification by column chromatography (silica gel 24 g, gradient 0 to 100% EtOAC in Heptane, 232 mg, 0.582 mmol, 62 %) as a yellow solid. A mixture of rotamers was observed by ¹H-NMR at room temperature. ¹H-NMR (DMSO, 600 MHz): δ 7.09 and 6.97 (2H, s), 3.68-3.63 (2H, m), 3.57-3.52 (2H, m), 3.20-3.14 (2H, m), 3.40-3.36 (2H, m), 1.31 (9H, s), 0.85 (9H, s), 0.02 (6H, s). LC/MS (Method A): MH+ 399.4, 1.39 min.

1-(2-((2-Hydroxyethyl)amino)ethyl)-1 H-pyrrole-2,5-dione (Linker 1 for ADC-1)

Under N₂: tert-butyl (2-((tert-butyldimethylsilyl)oxy)ethyl)(2-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl)ethyl)carbamate (220 mg, 0.552 mmol) was dissolved in 20 mL of DCM, and TFA (2.126 mL, 27.6 mmol) was added. Reaction mixture was stirred at room temperature for 2 h. Reaction mixture was concentrated in-vacuo, redissolved in a mixture of acetonitrile and water and freeze-dried to yield the desired product (185 mg, 0.552 mmol, 100 % yield) as a yellow oil as a trifluoroacetate salt. Linker 1 was used without further purification. ¹H-NMR (CD₃OD, 400 MHz): ¹H-NMR (CD₃OD, 400 MHz): δ 6.94 (2H, s), 3.92-3.87 (2H, m), 3.83-3.79 (2H, m), 3.32-3.28 (2H, m), 3.23-3.18 (2H, m).

Synthesis of Linker 2

a: ethanolamine, 90% yield; b: di-tert-butyl dicarbonate, TEA, THF, 50% yield;

c: maleimide, DIAD, PPh₃, 43% yield, d: CF₃COOH, 60% yield. tert-Butyl 3-((2-hydroxyethyl)amino)propanoate

Obtained as described in Aebi, Johannes; Binggeli, Alfred; Green, Luke; Hartmann, Guido; Maerki, Hans P.; Mattei, Patrizio; Ricklin, Fabienne; Roche, Olivier, Patent: US2010/16282 A1 , 2010 ; P. 23 tert-Butyl 3-((tert-butoxycarbonyl)(2-hydroxyethyl)amino)propanoate

Under N₂, tert-Butyl 3-((2-hydroxyethyl)amino)propanoate (1304 mg, 6.89 mmol) was dissolved in 20 mL of THF, and triethylamine (0.960 mL, 6.89 mmol), followed by Boc- anhydride (1.600 mL, 6.89 mmol) were added. Reaction mixture was stirred at room temperature for 4 hr. Reaction mixture was concentrated in-vacuo and partitioned between ethyl acetate and brine. Organics dried with Na₂S0₄, filtered and absorbed onto Isolute. The desired product was obtained after purification by column chromatography (silica gel 80 g, gradient 0 to 100% EtOAC in Heptane, 988 mg, 3.41 mmol, 50 %) as a colorless oil. tert-Butyl 3-((tert-butoxycarbonyl)(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)ethyl)amino)propanoate

Under N₂: To a stirred at -78°C solution of DIAD (0.863 mL, 4.44 mmol) and triphenylphosphine (1 164 mg, 4.44 mmol) in 10 mL of toluene a freshly prepared solution of maleimide (431 mg, 4.44 mmol) and tert-Butyl 3-((tert-butoxycarbonyl)(2- hydroxyethyl)amino)propanoate (988 mg, 3.41 mmol) in 10 mL of THF was added. Reaction mixture was allowed to warm up to room temperature and stirred overnight. Reaction mixture was diluted with DCM and washed with water. Organics dried with Na₂S0₄, filtered and absorbed onto Isolute. The desired product was obtained after purification by column chromatography (silica gel 80 g, gradient 0 to 100% EtOAC in Heptane, 903 mg, 1.48 mmol, 43 %, purity 60% by LC-MS, UV) as a colorless oil. LC/MS (Method A): MH+ 369.3, 1.11 min. tert-Butyl 3-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)propanoate

Under N₂: tert-Butyl 3-((tert-butoxycarbonyl)(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)ethyl)amino)propanoate (870 mg, 2.361 mmol) was dissolved in 5 mL of DCM and reaction mixture was cooled to -10 C. TFA (5 mL, 64.9 mmol) was added. Reaction mixture was stirred at -10°C for 3 hr, then concentrated in-vacuo, redissolved in in a mixture of acetonitrile and water and freeze-dried to yield 1086 mg of the crude desired product (1.420 mmol, 60.1 % yield, about 50% pure as determined by purification of a small batch) as a yellow oil.

100 mg of the crude compound was purified by reverse phase column chromatography to yield 50 mg of the pure Linker 2 (as a trifluoroacetate salt).

¹H-NMR (CDCI₃, 400 MHz): δ 6.76 (2H, s), 3.96-3.91 (2H, m), 3.39-3.29 (4H, m), 2.79- 2.73 (2H, m), 1.48 (9H, s). LC/MS (Method A): MH+ 269.6, 0.48 min.

Synthesis of Linker 4

tert-butyl 3-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)methyl)azetidine-1-carboxylate

To a solution triphenylphosphine ( 1 .40 g, 5.34 mmol) in dry THF (53 ml) at -78 °C was added dropwise over 5 mins diisopropyl azodicarboxylate (1 .04 ml, 5.34 mmol) and the resulting mixture was stirred for 5 mins. Tert-butyl 3-(hydroxymethyl)azetidine-1 - carboxylate (1 .00 g, 5.34 mmol) was then added to the reaction over 5 mins. The resulted solution was stirred for an additonal 5 mins when maleimide (0.518 g, 5.34 mmol) was added. The reaction was mixture was allowed to warm to RT and stirred for an additional 18h. The reaction was concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 44% yield; UPLC-MS: Rt = 0.87 mins; MS m/z [M+H]⁺ 267.0; Method A.

3-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)azetidin-1 -ium 2,2,2-trifluoroacetate

To a solution of tert-butyl 3-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)azetidine-1 - carboxylate (624 mg, 2.34 mmol) in DCM (23.5 ml) was added slowly trifluoroacetic acid (9.03 ml, 1 17 mmol) and the reaction mixture was stirred at RT for 30 mins. The reaction was concentrated to dryness to give the desired product as pale yellow solid in 99% yield; UPLC-MS: Rt = 0.22 mins; MS m/z [M+H]⁺ 167.0; Method A.

Synthesis of Linker 5

tert-butyl (3-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)amino)-3-oxopropyl)carbamate

To a solution of 3-tert-butoxycarbonyl)amino)propanoic acid (500 mg, 1 .97 mmol) and HATU (1 .50 g, 3.93 mmol) in DMF (20 ml) was added slowly triethylamine (1 .37 ml, 9.84 mmol) followed by 1 -(2-aminoethyl)-1 H-pyrrole-2,5-dione (500 mg, 1 .97 mmol) and the reaction mixture was stirred at RT for 24 h. The reaction was diluted EtOAc and washed with 1 M aqueous HCI solution. The organic layer was extracted, washed with saturated aqueous NaHC0₃ solution. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 24% yield; UPLC-MS: Rt = 0.65 mins; MS m/z [M+H]⁺ 312.1 ; Method A.

3-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)amino)-3-oxopropan-1 -aminium 2,2,2- trifluoroacetate

To a solution of tert-butyl 3-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)azetidine-1 - carboxylate (624 mg, 2.34 mmol) in DCM (23.5 ml) was added slowly trifluoroacetic acid (0.80 ml, 10.4 mmol) and the reaction mixture was stirred at RT for 30 mins. The reaction was concentrated to dryness to give the desired product as pale yellow solid in quantitative yield; UPLC-MS: Rt = 0.24 mins; MS m/z [M+H]⁺ 212.1 ; Method A.

2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl (4-nitrophenyl) carbonate

To a solution of 1-(2-hydroxyethyl)-1 H-pyrrole-2,5-dione (250 mg, 1.77 mmol) in DCM (8.9 ml) was added diisopropylethylamine (1.5 ml, 8.86 mmol) and bis(4-nitrophenyl) carbonate (701 mg, 2.30 mmol) and the reaction mixture was stirred at RT for 3h. The reaction was extracted with water and DCM. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as a pale yellow solid in 88% yield; H-NMR (DMSO, 400 MHz): δ 8.34-8.32 (2H, m), 7.54-7.51 (2H, m), 7.09 (2H, s), 4.36-4.34 (2H, m), 3.81-3.79 (2H, m).

1 -(2-(2-hydroxyethoxy)ethyl)-1 H-pyrrole-2,5-dione

To a solution of (2-aminoethoxy)ethanol (2.9 ml, 29.0 mmol) in a saturated aqueous NaHC0₃ soltuion (150 ml) was added at 0°C, N-(methoxycarbonyl)maleimide (4.5 g, 29.0 mmol) and the reaction mixture was stirred at RT for 30mins and then an additional 3h at RT. The reaction was extracted with DCM. The organic layers were combined, dried over Na₂SC>4, filtered and concentrated to dryness afforded the title compound as a pale yellow oil in 53% yield; UPLC-MS: Rt = 0.35 mins; MS m/z [M+H]⁺ 186.0; Method E. 2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl (4-nitrophenyl) carbonate

The product was synthesized in an analogous way as 2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)ethyl (4-nitrophenyl) carbonate using 1 -(2(2-hydroxyethoxy)ethyl-1 H)-pyrrole-2,5- dione instead; 63% yield; UPLC-MS: Rt = 1 .80 mins; MS m/z [M+H]⁺ 697.0; Method E.

Sulfonate-Substituted Linker Synthesis

The general method for making this linker is adapted from a published method, J. Med. Chem. 2011 , vol. 54, 3606-23; a pentafluorophenyl ester was selected instead of the N- hydroxysuccinimide used in the reference.

Sigma-Aldrich

1. ₂S₂0₅, H₂0, Heat

Synthesis Example 1. (3R.4R)-benzyl 3-(((S)-2-acetoxy-N-((RH1-benzyl-4-(2.5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)propanam

4-fluoropyrrolidine-1-carboxylate.

. LC/MS (uplc): MH+ 733.3, 1.36 min (Method A).

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4- yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hvdroxypropanamide

(General Method for Cbz-deprotection)

To a solution of (3R,4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (1 15 mg, 0.14 mmol) in MeOH (2 mL) was added Pd/C (30.4 mg, 0.03 mmol) and ammonium formate (108 mg, 1.7 mmol). The reaction mixture was heated at 55 °C for 1 h. Upon completion the reaction was filtered to remove Pd/C and solvent evaporated under reduce pressure to yield crude (S)-1-(((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(te^^

fluoropyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate : LC/MS (uplc): MH+ 599.2, 0.92 min. Crude was used without any further purification in the next step.

To a solution of (S)-1 -(((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate, from the Cbz-deprotection step, in MeOH (3 ml_) was added K₂C0₃ (197 mg, 1 .4 mmol). The reaction was stirred at room temperature for 1 h. Upon completion of the reaction by LC/MS (uplc, Method A), the crude mixture was filtered to remove the solids, and the desire product, (S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamide, was obtained upon purification by reverse phase column chromatography (PrepLC Method C), 60 mg, 0.09 mmol, 63 %). The product was isolated as the TFA salt. ¹H-NMR (DMSO, 600 MHz): δ 7.80 (1 H, m), 7.75 (1 H, m), 7.45-.7.25 (6H, m), 7.09 (1 H, m), 5.71 (1 H, m), 5.25 (2H, m), 5.1 1 (1 H, m), 4.95 (1 H, m), 4.05 (1 H, m), 3.80 (1 H, m), 3.35 (2H, m), 3.20 (1 H, m), 2.90 (1 H, m), 2.83 (1 H, m), 2.73 (1 H, m), 2.68 (1 H, m), 2.22 (1 H, m), 1 .87 (1 H, m), 1 .45 (1 H, m), 1 .35 (1 H, m), 1 .25 (3H, m), 1 .09 (1 H, m), 0.67 (1 H, m). Missing signals hidden under solvent peak. LC/MS (upl): MH+ 557.2, 0.84 min (Method A).

General Method for BOC Protection

To a solution of the urea payload (1 mmol) in MeOH (0.1 M), were added K₂C0₃ (2 mmol) and Boc anhydride (3 mmol). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was filtered to remove solids, and the desire product Boc-urea was isolated after purification by normal phase column chromatography (PrepLC Method A or B).

(3R.4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2.5-difluorophenvn-1 H-imidazol-2- yl)(tetrahvdro-2H-pyran-4-yl)methyl)-2-hvdroxypropanamido)methyl)-4-fluoropyrrolidine-1 - carboxylate.

LC/MS (uplc): MH+ 657.3, 1.30 min (Method A).

Synthesis Example 2. (S)-2-amino-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-

2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3- hydroxypropanamide

General Procedure for Cbz-deprotection.18 mg, 0.023 mmol, 49%.¹H-NMR (DMSO, 600 MHz): δ 7.80 (1H, m), 7.75 (1H, m), 7.40 (2H, m), 7.32 (4H, m), 7.11 (1H, m), 5.27 (1H, m), 5.29 (1H, m), 5.14 (1H, m), 4.98 (1H, m), 3.82 (1H, m), 3.79 (1H, m), 3.65 (1H, m), 3.51 (1H, m), 3.40 (2H, m), 2.92 (1H, m), 2.84 (1H, m), 2.78 (1H, m), 2.59 (1H, m), 2.20 (1H, m), 2.01 (1H, m), 1.88 (1H, m), 1.65 (1H, m), 1.45 (1H, m), 1.30 (1H, m), 1.18 (1H, m), 0.89 (1H, m), 0.65 (1H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 572.2, 0.71 min (Method A).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butoxycarbonyl)amino)-3- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate.

General Procedure for Boc Protection: LC/MS (uplc): MH+ 772.2, 1.36 min (Method A).

Synthesis Example 3. (3R,4R)-benzyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3-((S)-1-hvdroxypropan-2- yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate

792 mg, 1.0 mmol, 76%.¹H-NMR (DMSO, 600 MHz): δ 7.70 (1 H, m), 7.52 (1 H, m), 7.30- 7.10 (10H, m), 6.90 (1 H, m), 5.78 (s, 1 H), 5.37 (2H, bs), 5.34 (2H, m), 5.30 (1 H, bs), 5.05 (1 H, m), 4.92 (2H, bs), 4.25 (2H, bs), 3.87 (2H, m), 3.81 (1 H, m), 3.71 (1 H, m), 3.64 (2H, m), 3,43 (2H, m), 3.26 (1 H, m), 2.61 (1 H, m), 2.45 (1 H, m), 2.01 (1 H, m), 1.61-1.25 (4H, m), 1.12 (3H, m). LC/MS (uplc): MH+ 720.3, 1.25 min. (Method A).

Synthesis Example 4. (3R.4R)-benzyl 3-(((3S.4R)-N-((R)-(1-benzyl-4-(2.5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3,4- dihvdroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate

2870 mg, 2.3 mmol, 60%. LC/MS (uplc): MH+ 748.2, 1.19 min (Method A).

Synthesis Example 5. (3R,4R)-benzyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3-(2,3-dihvdroxypropyl)ureido)methyl)-4- fluoropyrrolidine-1-carboxylate

670mg, 0.9 mmol, 39%. LC/MS (uplc): MH+ 736.2, 1.16 min. (Method A).

(3R,4R)-benzyl 3-((N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)-3-hydroxyazetidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- carboxylate.

572 mg, 0.64 mmol, 66%. LC/MS (uplc): MH+ 718.2, 1.23 min (Method A).

(3R,4R)-benzyl 3-((N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)-3-hydroxypiperidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- carboxylate.

Isomer A: LC/MS (uplc): MH+ 746.2, 1.27 min (Method A).

Isomer B: LC/MS (uplc): MH+ 746.2, 1.28 min (Method A). Cbz Deprotection:

To a solution of Cbz-Urea Payload (1.0 mmol) in MeOH (0.1 M), was added Pd/C (content 10%, 0.2 mmol) and ammonium formate (12 mmol). The reaction was heated at 55 °C for 30 min. Upon completion the reaction was filtered to remove Pd/C and the desired urea isolated upon reverse phase column chromatography. (PrepLC Method C or

D). Synthesis Example 6. 1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- yl)(tetrahvdro-2H-pyran-4-yl)methyl)-1-(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)-3-((S hvdroxypropan-2-yl)urea

¹H-NMR (DMSO, 600 MHz): δ 7.75 (2H, m), 7.40-7.25 (6H, m), 7.09 (1H, m), 5.90 (1H, bs), 5.36-5.30 (3H, m), 4.96 (1H, m), 4.71 (1H, m), 3.85 (2H, m), 3.79 (1H, m), 3.58 (2H, m), 3.29 (4H, m), 2.79 (2H, m), 2.57 (1H, bs), 2.17 (1H, bs), 1.72 (1H, m), 1.60 (1H, m), 1.38 (2H, m), 0.95 (2H, m), 1.08 (3H, bs). 1 signal hidden under the solvent peak. LC/MS (uplc): MH+ 586.3, 0.86 min. (Method A).

1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-(2-hydroxyethyl)urea.

15 mg, 0.025 mmol, 19%.¹H-NMR (DMSO, 600 MHz): 57.78-7.68 (2H, m), 7.44-7.36 (2H, m), 7.36-7.25 (4H, m), 7.15-7.04 (1H, m), 6.45-6.28 (1H, m), 5.45-5.21 (3H, m), 5.10-4.91 (1H, m), 4.71-4.57 (1H, m), 3.91-3.79 (1H, m), 3.67-3.52 (2H, m), 3.28-3.14 (4H, m), 3.09- 2.91 (1H, m), 2.84-2.68 (1H, m), 2.24-2.12 (1H, m), 1.97-1.78 (1H, m), 1.59-1.49 (1H, m), 1.47-1.37 (1H, m), 1.36-1.27 (1H, m), 1.22-1.10 (1H, m), 0.79-0.60 (1H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 572.2, 0.83 min (Method A).

Synthesis Example 7. (3S,4R)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- yl)(tetrahvdro-2H-pyran-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3,4- dihvdroxypyrrolidine-1-carboxamide

¹ H-NMR (DMSO, 600 MHz): δ 7.73 (2H, bs), 7.38-7.25 (6H, m), 7.06 (1H, bs), 5.5 (2H, m), 4.95 (1H, m), 4.84 (2H, bs), 4.82 (1H, bs), 4.02 (2H, m), 3.81 (1H, m), 3.60 (1H, bs), 3.49 (2H, bs), 3.27 (1H, m), 3.13 (1H, bs), 2.93 (1H, m), 2.72 (1H, m), 2.39 (1H, bs), 2.28 (1H, m), 1.85 (1H, bs), 1.73 (1H, m), 1.58 (1H, m), 1.03 (1H, m), 0.97 (1H, m), 0.28 (1H, m), 5 H missing hidden under solvent peak. LC/MS (uplc): MH+ 614.3, 0.82 min. (Method A).

Synthesis Example 8. 1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2- yl)(tetrahvdro-2H-pyran-4-vnmethyl)-3-(2.3-dihvdroxypropyn-1-(((3S.4R)-4- fluoropyrrolidin-3-yl)methyl)urea

¹H-NMR (DMSO, 600 MHz): δ 7.76 (2H, bs), 7.40-7.25 (6H, m), 7.09 (1H, bs), 6.36 (1H, bs), 5.40 (1H, bs), 5.30 (2H, m), 4.95 (1H, m), 4.79 (1H, bs), 4.60 (1H, bs), 3.84 (1H, m), 3.61 (1H, m), 3.56 (1H, m), 3.54 (1H, bs), 3.29 (1H, m), 3.25 (4H, m), 3.10 (1H, m), 2.94(1H, m), 2.68 (1H, m), 2.56 (1H, m), 2.13 (1H, m), 1.80 (1H, m), 1.49 (1H, m), 1.42 (1H, m), 1.35 (1H, m), 1.18 (1H, m), 0.71 (1H, m).2 signals hidden under the solvent peak. LC/MS (uplc): MH+ 602.3, 0.78 min. (Method A).

N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-hydroxyazetidine-1-carboxamide.

45 mg, 0.073 mmol, 92%. LC/MS (uplc): MH+ 584.2, 0.84 min (Method A).

N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-hydroxypiperidine-1-carboxamide.

Isomer A: LC/MS(uplc): MH+ 612.30.88 min (Method A).

Isomer B: LC/MS(uplc): MH+ 612.30.89 min (Method A). Synthesis Example 9. (3R.4R)-tert-butyl 3-((1 -((R)-(1-benzyl-4-(2.5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3-((S)-1-hvdroxypropan-2- yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate

General Procedure for the Synthesis of Boc-Urea Payloads:

Urea Coupling: To an ice-cooled solution of phosgene (20% in toluene, 2 mmol) in CH₂CI₂ (0.1 M) was added a solution of the amine (1 mmol) and triethylamine (3 mmol) in CH₂CI₂ (1 M). The reaction mixture was stirred at room temperature for 30 min. Upon completion of the reaction by LC/MS (uplc, Method A), the desire amine for the urea (20 mmol) was added, and the reaction stirred at 60 °C for 2 h, and then, at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude solvent was evaporated under reduce pressure, and the desire product was obtained after purification by normal phase column chromatography (PrepLC Method A or B).

0.49 mg, 0.69 mmol, 68%. LC/MS (uplc): MH+ 686.3, 1.25 min. (Method A).

(3R,4R)-tert-butyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)-3-(2-hydroxyethyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate.

1.05 g, 1.6 mmol, 85 %. LC/MS (uplc): MH+ 782.2, 0.90 min (Method A).

Synthesis Example 10. (3R.4R)-tert-butyl 3-(((3S.4R)-N-((R)-(1-benzyl-4-(2.5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3,4- dihvdroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate

817 mg, 1.09 mmol, 47%. LC/MS (uplc): MH+ 714.2 , 1.18 min. (Method A).

Synthesis Example 11. (3R.4R)-tert-butyl 3-((1-((R)-(1-benzyl-4-(2.5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)-3-(2,3-dihvdroxypropyl)ureido)methyl)-4- fluoropyrrolidine-1-carboxylate

317 mg, 0.43 mmol, 49%. LC/MS (uplc): MH+ 702.2, 1.16 min. (Method A).

(3R,4R)-tert-butyl 3-((N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-

2H-pyran-4-yl)methyl)-3-hydroxyazetidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- carboxylate.

240 mg, 0.33 mmol, 73%. LC/MS (uplc): MH+ 684.2, 1.23 min (Method A).

2H-pyran-4-yl)methyl)-3-hydroxypiperidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1- carboxylate.

Isomer A: LC/MS (uplc): MH+ 712.3, 1.29 min (Method A).

Isomer B: LC/MS (upcl): MH+ 712.3, 1.30 min (Method A).

Synthesis Example 12. (3R.4RHert-butyl 3-((3-((S)-1-azidoDropan-2-vn-1-((R)-(1- benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4- yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate

To an ice-cooled solution of (3R,4R)-tert-butyl 3-((1-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-((S)-1- hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate (100 mg, 0.15 mmol) in CH₂CI₂ (0.8 mL) and pyridine (24 pL, 0.29 mmol), was slowly added p-tosyl chloride (40.3 mg, 0.21 mmol). The reaction mixture was stirred at room temperature. Upon completion of the reaction, the reaction mixture was diluted in CH₂CI₂ and partitioned between H₂0 and CH₂CI_2. The organic layer was separated and washed with H₂0 twice, dried over Na₂S0₄, filtered and evaporated under reduce pressure to give crude (3R,4R)- tert-butyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-3-((S)-1-(tosyloxy)propan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1- carboxylate (109 mg, 0.1 mmol, 71 %). The crude mixture was used without further purification. LC/MS (uplc): 704.3 (-135). (Method A).

To a solution of (3R,4R)-tert-butyl 3-((1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-((S)-1 -(tosyloxy)propan-2-yl)ureido)methyl)-4- fluoropyrrolidine-1 -carboxylate (109 mg, 0.1 mmol) in DMF (0.6 ml_), was added sodium azide and the reaction mixture stirred at 70 °C. Upon completion of the reaction, the mixture was cooled down to room temperature and diluted in EtOAc (5ml_). The reaction mixture was partitioned between H₂0 and EtOAc. The organic layer was separated and washed with H₂0 twice, dried over Na₂S0₄, filtered and solvent evaporated under reduce pressure. The desire product (3R,4R)-tert-butyl 3-((3-((S)-1 -azidopropan-2-yl)-1 -((R)-(1 - benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1 -carboxylate was obtained after purification by column chromatography (gradient 30 to 100% EtOAC in Heptane, 40 mg, 0.053 mmol, 52%). ¹H-NMR (DMSO, 600 MHz): δ 7.73 (1 H, bs), 7.68 (1 H, bs), 7.36-7.29 (6H, m), 7.09 (1 H,bs), 6.29 (1 H, bs), 5.34 (2H, m), 5.07 (1 H, m), 3.97 (1 H, m), 3.85 (1 H, m), 3.70 (2H, m), 3.50 (1 H, m), 3.37-3.20 (6H, m), 2.69 (1 H, m), 2.58 (1 H, m), 2.17 (1 H, m), 1 .96 (1 H, m), 1 .45 (1 H, m), 1 .30 (1 H, m), 1 .18 (9H, s), 1 .12 (1 H, m), 0.86 (2H, m). 3 signals hidden under the solvent peak. LC/MS (uplc): MH+ 71 1 .4, 1 .41 min. (Method A).

(3R.4R)-tert-butyl 3-((3-((S)-1 -aminopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2.5-difluorophenvn-

1 H-imidazol-2-yl)(tetrahvdro-2H-pyran-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1 - carboxylate

To a solution of (3R,4R)-tert-butyl 3-((3-((S)-1-azidopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4- fluoropyrrolidine-1 -carboxylate (100 mg, 0.14 mmol) in THF (2 ml_), was added triphenylphosphine (1 1 1 mg, 0.42 mmol) and H₂0 (51 μΙ_, 2.81 mmol). The reaction mixture was stirred at 50 °C. Upon completion of the reaction, the mixture was cooled down to room temperature, and diluted with EtOAc (5 mL). The reaction mixture was partitioned between EtOAc and H₂0. The organic layer was washed with H₂0, dried over Na₂S0₄, filtered and solvent evaporated under reduce pressure. The desire product (3R,4R)-tert-butyl 3-((3-((S)-1 -aminopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-fluoropyrrolidine-1 - carboxylate was isolated after column chromatography (gradient of 0 to 10% MeOH in CH₂CI₂, 51 mg, 0.071 mmol, 50%). LC/MS (uplc): MH+ 685.4, 1 .08 min. (Method A).

3-((S)-1 -aminopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahvdro-2H-pyran-4-yl)methyl)-1 -(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea

To a solution of (3R,4R)-tert-butyl 3-((3-((S)-1 -aminopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4- fluoropyrrolidine-1 -carboxylate (40 mg, 0.058 mmol) in Acetonitrile (1 .2 mL), trifluoroacetic acid (0.6 mL) was added and the reaction mixture stirred at room temperature. Upon completion of the reaction, the crude was filtered to remove the solids and the desire product 3-((S)-1 -aminopropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-1 -(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea was isolated after reverse phase column chromatography (gradient 5% to 35 % of MeCN (+0.1 % TFA) in H₂0 (+0.1 % TFA), 19.1 mg, 0.031 mmol, 53%). The TFA salt was neutralized with PL-HC03 MP SPE columns to afford the free base. ¹H-NMR (DMSO, 600 MHz): δ 7.75 (2H, bs), 7.40-7.28 (6H, m), 7.09 (1 H, bs), 6.02 (1 H, bs), 5.40-5.30 (3H, m), 4.95 (1 H, m), 3.84 (1 H, m), 3.74 (1 H, m), 3.61 (2H, m), 3.34 (2H, m), 3.24 (2H, m), 2.95 (1H, m), 2.72 (1H, m), 2.66 (1H, m), 2.59 (1H, m), 2.55 (1H, m), 2.19 (1H, m), 1.82 (1H, m), 1.56 (1H, m), 1.42 (1H, m), 1.34 (1H, m), 1.119 (1H, m), 1.08 (3H, bs), 0.71 (1H, m).2 signals hidden under the solvent peak. LC/MS (uplc): MH+ 585.3, 0.73 min. (Method A).

Synthesis Example 13

(R)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylpent-4-enoic acid^' Prepared as described in WO2005/54186 A2, 2005; P.48-49

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-3,3- dimethylpent-4-enoate

Under N₂: To an ice-cooled solution of 2-Chloro-1 -(2,5-difluorophenyl)ethanone (1 .974 g, 10.36 mmol) and K₂C0₃ (1 .074 g, 7.77 mmol) in 150 mL of acetone was added (R)-2- ((tert-butoxycarbonyl)amino)-3,3-dimethylpent-4-enoic acid (2.1 g, 8.63 mmol) followed by Kl (0.358 g, 2.158 mmol), cooling bath was removed and the reaction mixture was stirred at RT for 3.5 h.

The reaction mixture was poured onto crushed ice and extracted with DCM. Organics dried with Na₂S0₄, filtered and absorbed onto Isolute. The desired product was obtained after purification by column chromatography (silica gel 80 g, gradient 0 to 20% EtOAC in Heptane, 2.95 g, 86 %) as a yellow solid. ¹ H-NMR (DMSO, 400 MHz): δ 7.72-7.57 (2H, m), 7.55-7.45 (1 H, m), 6.94 (1 H, d, 8.9 Hz), 5.97 (1 H, dd, 17.4, 10.7 Hz), 5.40-5.25 (2H, m), 5.10-4.95 (2H, m), 4.1 1 (1 H, d, 9.0 Hz), 1 .39 (9H, s), 1 .13 (6H, s). LC/MS (Method C): MH+ 398.2, 3.25 min.

(R)-tert-butyl (1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)carbamate

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-3,3-dimethylpent-4- enoate (2.95 g, 7.42 mmol) was dissolved in 45 mL of toluene and ammonium acetate (1 1 .44 g, 148 mmol) was added. The resulting mixture was heated to reflux for 40 h.

The reaction mixture was cooled to RT, washed with water, sat. aq. NaHC0₃ and brine, dried with Na₂S0₄, filtered and concentrated in-vacuo. Residue was dried under reduced pressure at 40°C for 42 h. (2.69 g, 93 %, 97% pure by LC-MS, UV) as a yellow foam and used in the next step without further purification. LC/MS (Method A): MH+ 378.5, 1.22 min.

(R)-tert-butyl (1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3- en-1 -yl)carbamate

To a solution of (R)-tert-butyl (1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut- 3-en-1-yl)carbamate (2.69 g, 6.91 mmol) in 20 mL of DMF at 0 C was added K₂C0₃ (1.91 1 g, 13.83 mmol), followed by benzyl bromide (0.904 mL, 7.6 mmol). The resulting mixture was stirred at RT for 3 h.

Ice water was added, causing a precipitate. The off-white solid was collected by filtration, washed with DMF/water (1/2), water and dried under reduced pressure for 48 h to yield the desired product (2.9 g, 90 %) as a solid and used in the next step without further purification. LC/MS (Method A): MH+ 468.2, 1.53 min.

(R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- amine

Under N₂: (R)-tert-butyl (1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1-yl)carbamate (10.768 g, 23.03 mmol) was dissolved in 180 mL of DCM and reaction mixture was cooled to 0°C, then TFA (44.4 ml, 576 mmol) was added dropwise. Reaction mixture was stirred at 0 C for 5 min, then at RT for 1 h. Residue was concentrated under reduced pressure, diluted with DCM and basified with NaOH (2M). Extracted with DCM (3x), organics dried with Na₂S0₄, filtered and concentrated. The desired product (8.7 g, 23.68 mmol, 100 % yield, 97% pure) were obtained as a yellowish solid and used in the next step without any purification. LC/MS (Method A): MH+ 368.3, 0.90 min.

(3R,4R)-tert-butyl 3-((((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate

Under N2: To a solution of (3R,4S)-tert-butyl 3-fluoro-4-(hydroxymethyl)pyrrolidine-1- carboxylate (4.07 g, 18.55 mmol) in CH₂CI₂ (120 mL) was added Dess-Martin periodinane (13.1 1 g, 30.9 mmol). The reaction mixture was stirred at room temperature for 30 min. Upon completion of the reaction, the crude, (3R,4S)- tert-butyl 3-fluoro-4- formylpyrrolidine-1 -carboxylate was used as a solution in the next step without further treatment.

To a solution of (R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut- 3-en-1 -amine (5.68 g, 15.46 mmol), sodium triacetoxyborohydride (16.38 g, 77 mmol) and molecular sieves (20 g) in CH₂CI₂ (120 mL) at 0°C was added the solution of (3R,4S)- tert-butyl 3-fluoro-4-formylpyrrolidine-1-carboxylate in CH₂CI₂ from the previous step. The reaction mixture was stirred at RT for 1 h. The reaction mixture was filtered, diluted with DCM and washed with sat. NaHC0₃ and brine. Organics were dried with Na₂S0₄, filtered and absorbed onto Isolute. The desired product was obtained after purification by column chromatography (330 g silica gel, 0 to 40 % EtOAc in Heptane, 3.186 g, 5.6 mmol, 36%). LC/MS (Method B): MH+ 569.3, 6.60 min.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)propanamido)methyl)-4-fluoropyrrolidine-1- carboxylate

Under N₂: (3R,4R)-tert-butyl 3-((((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate (3.03 g, 5.33 mmol) in CH₂CI₂ (50 mL) at 0 C was added DIPEA (4.65 ml, 26.6 mmol) followed by (S)- 2-acetoxy propionyl chloride (1.349 ml, 10.66 mmol). The resulting solution was stirred at 0°C for 5min then warmed up to RT and stirred for 2h. Reaction mixture was diluted with DCM and washed with sat. NaHC0₃ then sat. NaCI. The organic layer was dried, concentrated and absorbed onto isolute. The desired product was obtained after purification by column chromatography (120 g silica gel, 0 to 50 % EtOAc in Heptane, 3.143 g, 4.6 mmol, 86%) as a colorless solid. LC/MS (Method A): MH+ 683.3, 1.51 min.

Synthesis Example 14.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

Under N₂: A solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1 -yl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate (1 .5 g, 2.197 mmol) in THF (20 mL) was cooled to 0°C and a solution of borane in THF (4.39 mL, 4.39 mmol, 1 M) was added dropwise. Reaction mixture was allowed to warm up to RT and stirred for 4h. It was cooled to 0°C and quenched with 25 mL of THF/EtOH 1 : 1 , followed by 40 mL of phosphate buffer (pH 7), finally H₂0₂ (2.244 mL, 21 .97 mmol, 30% aq) was added and reaction mixture was stirred at RT overnight. Brine was added to the reaction mixture and it was extracted 3 times with ETOAc. Organic layer was washed successively 3 times with cold saturated Na₂S₂0₅, water and brine, dried with Na₂S0₄, filtered and absorbed onto isolute. The desired product was obtained after purification by column chromatography (80 g silica gel, 0 to 100 % EtOAc in Heptane, 1 .075 g, 1 .519 mmol, 69%) as a colorless oil. LC/MS (Method A): MH+ 701 .4, 1 .33 min. A mixture of rotamers by ¹H-NMR at room temperature. ¹H- NMR (DMSO, 400 MHz): δ 7.85-7.65 (2H, m), 7.41 -7.28 (6H, m), 7.15-7.05 (1 H, m), 5.86 and 5.81 (1 H, two singlets, rotamers), 5.40-5.17 (3H, m), 4.96 (1 H, d, 15 Hz), 4.22-4.14 (1 H, m), 3.95-3.70 (2H, m), 3.27-3.20 (1 H, m), 3.15-2.97 (1 H, m), 2.63-2.55 (1 H, m), 2.22- 2.14 (1 H, m), 2.1 1 (3H, s), 1 .70-1 .49 (5H, m), 1 .35-1 .20 (3H, m), 1 .09 (9H, s), 0.95 (6H, s).

(S)-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 16 h) after purification by reverse phase column chromatography (17 mg, 0.024 mmol, 85% yield, as a TFA salt).

LC/MS (Method B): [M+H]⁺ 559.3; Rt 3.25 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, ratio about 3:1 ): δ 9.01 (1 H, br s), 8.69 (1 H, br s), 8.00 and 7.92 (1 H, two dublets for two rotamers 3.6 and 4.2Hz), 7.45-7.30 (7H, m), 7.17-7.07 (1 H, m), 5.87 and 5.85 (1 H, two singlets for two rotamers), 5.50-5.00 (3H, m), 4.65-4.58 (1 H, m), 4.30-3.90 (2H, m), 3.35-3.15 (6H, m), 2.42-2.32 (1 H, m), 1.97-1.85 (2H, m), 1.55-1.40 (1 H, m), 1.40- 1.20 (4H, m), 1.02 and 0.90 (3H, two singlets for two rotamers), 0.82 and 0.68 (3H, two singlets for two rotamers).

Synthesis Example 15.

Step 1 : (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3,3- dimethylbutanoic acid

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-^

fluoropyrrolidine-1-carboxylate (200 mg, 0.285 mmol) in Acetone (Volume: 7 ml) at 0°C, Jones reagent (2M, 0.856 ml, 1.712 mmol) was added dropwise. The obtained yellow solution was stirred at room temperature for 2.5h.

The excess of Jones reagent was quenched with isopropanol (2ml) at 0°C dropwise, then the reaction mixture was concentrated, dilluted with water and extracted with ethyl acetate (^*3). The combined organic layers were dried and evaporated to afford the titled compound (198 mg, 0.255 mmol, 89% yield, 92% pure) which was used in the next step without any further purification.

LC/MS (Method A): [M+H]⁺ 715.3, Rt 1.27 min.

Step 2: (R)-4-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3R,4R)-1 - (tert-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanoic acid

In a stirring solution of (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imida 2-yl)-3,3-dimethylbutanoic acid (step 1 ) (928 mg, 1.078 mmol) in Methanol (Volume: 40 ml) was addded NaOH (1 M, 3.23 ml, 3.23 mmol) and the reaction mixture was stirred at room temperature for 1 h. Reaction mixture diluted with water, evaporated, acidified with HCI (1 M) and extracted with EA (^*3). The combined organic layers were dried and concentrated to give desired product (980 mg, 1.005 mmol, 93% yield, 69% pure) which was used in the next step without any further purification.

LC/MS (Method A): [M+H]⁺ 673.4, Rt 1.23 min.

Step 3: (3R,4R)-tert-butyl 3-(((S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-2,2-dimethyl-4-(methylamino)-4-oxobutyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3R,4R)-1-(tert- butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanoic acid (step 2) (38 mg, 0.056 mmol) and 2 M solution of Methylamine in THF (0.1 13 ml, 0.226 mmol) were dissolved in DMF (Volume: 2 ml) and DIPEA (0.049 ml, 0.282 mmol), followed by HATU (32.2 mg, 0.085 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. Diluted with EA and washed with brine (^*3). The combined organic layers were dried and concentrated to give 37mg of desired product (37 mg, 0.026 mmol, 46.8 % yield, 49% pure) which was used in the next step without any further purification.

LC/MS (Method A): [M+H]⁺ 686.3, Rt 1.22 min. Step 4: (R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-N,3,3-trimethylbutanamide

(3R,4R)-tert-butyl 3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethyl-4-(methylamino)-4-oxobutyl)-2-hydroxypropanamido)m

1-carboxylate (step 3) (37 mg, 0.026 mmol, 49% pure) was dissolved in Acetonitrile (Volume: 2 ml) and TFA (0.416 ml, 5.40 mmol) was added and the reaction mixture was stirred at rt for 1h.

Concentrated and purified by reverse phase chromatography to yield desired product (6.5 mg, 9.29 μιτιοΙ, 35 % yield as a TFA salt).

LC/MS (Method B): [M+H]⁺ 586.7, Rt 3.03 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers with the ratio about 5:1, some peaks for the minor rotamer can't be clearly identified): δ 8.99 (1.2H, br s), 8.71 (1.2H, br s), 7.91-7.86 (0.2H, m), 7.84-7.81 (1H, m), 7.80-7.75 (1H, m), 7.69-7.65 (1H, m), 7.57 (0.2H, d, 3.6 Hz), 7.45-7.29 (6.5H, m), 7.15- 7.10 (1.4H, m), 6.22 (1H, s), 5.78 (0.2H, s), 5.59 (0.2H, d, 16.2 Hz), 5.48 (0.2H, d, 16.2 Hz), 5.34 (1H, d, 15.1 Hz), 5.17 (1H, d, 15.1 Hz), 5.10 (1H, d, 52.2 Hz), 4.83-4.77 (0.2H, m), 4.57-4.51 (1H, m), 4.49-4.42 (0.2H, m), 4.06-4.01 (0.4H, m), 3.95-3.92 (2H, m), 3.35- 3.24 (1.2H, m), 3.14-3.00 (1.2H, m), 2.56 (0.6H, d, 4.2 Hz), 2.31-2.23 (1H, m), 2.19 (1H, d, 13.5 Hz), 2.10-2.00 (0.4H, m), 2.00-1.92 (1H, m), 1.90 (1H, d, 13.5 Hz), 1.72-1.58 (1H, m), 1.35 (3H, d, 6.0 Hz), 1.15 (0.6H, s), 1.08 (0.6H, s), 1.05 (3H, s), 0.95 (3H, s), 0.77 (0.6H, d, 6.0 Hz). One CH₃ group is hidden under DMSO peak, OH not seen.

Synthesis Example 16,

Step 1 : (3R,4R)-tert-butyl 3-(((2S)-2-acetoxy-N-((1 R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-3,4-dihydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

In a stirring solution of 4-methylmorpholine-N-oxide (73.6 mg, 0.628 mmol) and osmium tetroxide 4% (0.082 ml_, 10.47 μιτιοΙ) in Water (6 mL) was added (3R,4R)-tert-butyl-3- (((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1 -yl)propanamido)methyl)-4-f luoropyrrolidine-1 -carboxylate ( 143 mg, 0.209 mmol) in THF (4 ml). Mixture was stirred at room temperature for 18h. Reaction mixture was quenched with saturated aquous Na2S2C>5 and it was stirred 1 h at room temperature, then extracted with DCM (3 times). Organic layer was dried on Na2SC>4, filtered and concentrated to give 157 mg of desired product (157 mg, 0.169 mmol, 81 % yield, 77% pure) which was used in the next step without any further purification. LC/MS (Method A): [M+H]⁺ 717.3; Rt 1.23/1.28 min. (2 diastereoisomers).

Step 2: (3R,4R)-tert-butyl-3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

(3R,4R)-tert-butyl-3-(((2S)-2-acetoxy-N-((1 R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-3,4-dihydroxy-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidi carboxylate (step 1 ) (120 mg, 0.129 mmol) and K₂C0₃ (35.6 mg, 0.258 mmol) were dissolved in THF (6ml_). Nal04 (83 mg, 0.387 mmol) in water (4 ml) was added. Mixture was stirred at room temperature for 3h. White precipitate was removed by filtration, then filtrate was extracted 3 times with EA. Organic layer was washed successively with saturated Na2S2C>5 then with brine, dried on Na2SC>4, filtered and concentrated. No further purification, 105 mg of desired product (105 mg, 0.090 mmol, 70% yield, 59% pure) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 685.4; Rt 1.42 min.

Step 3: (R)-3-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylpropanoic acid

Under N₂: In a solution of (3R,4R)-tert-butyl-3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (step 2) (102 mg, 0.149 mmol) in Acetone (10 mL) at 0°C, Jones reagent 2M (0.372 mL, 0.745 mmol) was added dropwise. The obtained yellow solution was stirred at room temperature for 3h. The excess of Jones reagent was quenched with isopropanol (6mL) at 0°C dropwise, then the reaction mixture was concentrated, dilluted with water and extracted with ethyl acetate (^*3). The combined organic layer were dried on Na2SC>4, filtered, and concentrated.

81.5 mg of desired product (81.5 mg, 0.078 mmol, 52% yield, 67% pure) were obtained and used to the next step without further purification.

LC/MS (Method A): [M+H]⁺ 701.3; Rt 1.29 min.

Step 4: (3R,4R)-tert-butyl-3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)propanamido)methyl)- 4-f I u o ro pyrrol i d i n e-1 -ca rboxy late

(R)-3-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-im^

dimethylpropanoic acid (step 3) (46 mg, 0.066 mmol) and Methylamine 2 M in THF (0.656 ml, 1.313 mmol) were dissolved in DMF (2 ml_). DIPEA (0.057 ml_, 0.328 mmol), followed by HATU (37.4 mg, 0.098 mmol) were added. Reaction mixture was stirred at room temperature for 24h. Mixture was diluted with ethyl acetate and washed with brine. Organic layer was dried on Na₂S0₄,filtered and concentrated to give 46.9 mg of desired product (46.9 mg, 0.066 mmol, 100% yield) which was used in the next step without any further purification.

LC/MS (Method A): [M+H]⁺ 714.3; Rt 1.29 min.

Step 5: (S)-1-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethyl- 3-(methylamino)-3-oxopropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1- oxopropan-2-yl acetate

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)propanamido)methyl)-4-fluoropyrrolidine- 1-carboxylate (step 4) (46.9 mg, 0.066 mmol) was dissolved in Acetonitrile ( 2 ml ) and water (1 ml). TFA (0.506 ml, 6.57 mmol) was added and the reaction mixture was stirred at 60^°C for 4h. Solution was diluted in EA, washed with saturated aquous NaHCC>3, then with brine. Organic layer was dried on Na2S04, filtered and concentrated. No further purification, 40.3 mg of desired product (40.3 mg, 0.066 mmol, 100% yield) were obtained and used in the next step.

LC/MS (Method A): [M+H]⁺ 614.3; Rt 0.89 min. Step 6: (R)-3-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-3-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-N,2,2-trimethylpropanamide

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-

2-yl)-2,2-dimethyl-3-(methylamino)-3-oxopropyl)propanamido)m

1-carboxylate (step 5) (40.3 mg, 0.066 mmol) was dissolved in MeOH (3 ml_). K₂C0₃

(45.4 mg, 0.328 mmol) was added. Reaction mixture was stirred at room temperature for

40 min.

Mixture was filtered and submitted to purification by reverse phase chromatography. 8.9 mg of expected product (8.9 mg, 0.012 mmol, 19% yield, 94% pure) were obtained as a TFA salt.

LC/MS (Method B): [M+H]⁺ 572.2; Rt 2.92 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks for the major rotamer are reported): δ 9.04 (1 H, br s), 8.85 (1 H, br s), 7.76-7.70 (3H, m), 7.45-7.31 (6H, m), 7.13-7.07 (1 H, m), 6.42 (1 H, s), 5.25-5.20 (2H, m), 5.14-5.09 (1 H, m), 4.57-4.51 (1 H, m), 3.92-3.85 (2H, m), 3.35-3.25 (1 H, m), 3.20-3.05 (1 H, m), 2.48 (3H, d, 4.5Hz), 2.33-2.25 (1 H, m), 1.98-1.88 (1 H, m), 1.82-1.68 (1 H, m), 1.36-1.32 (6H, m), 1.06 (3H, s).

Synthesis Example 17

Step 1 : (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)-3-hydroxy-2,2-dimethylpropyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethyl-3-oxopropyl)propanamido)methyl)-^^ fluoropyrrolidine-1 -carboxylate (232.6 mg, 0.340 mmol) in THF (12 ml) at 0 C under N₂ were added NaBH₄ (19.28 mg, 0.510 mmol) and 2 mL of MeOH to get a clear solution. Reaction mixture was stirred at room temperature for 1 h. Mixture was quenched at 0 C with 1 mL of saturated aquous NH4CI, extracted with EA, dried with Na2SC>4, filtered and concentrated.

No further purification, 233 mg of desired product (233 mg, 0.340 mmol, 100% yield, 100% pure) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 687.3; Rt 1.37 min

Step 2: (S)-1 -(((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3-hydroxy-2,2- dimethylpropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-3-hydroxy-2,2-dimethylpropyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (step 1 ) (26 mg, 0.038 mmol) was dissolved in Acetonitrile (1 mL) and water (0.5 mL).TFA (0.292 mL, 3.79 mmol) was added and it was stirred at 60^°C for 1 h. Reaction mixture was diluted in EA, washed with saturated NaHCC>3, then with brine. Organic layer was dried on Na2SC>4, filtered and concentrated.

No further purification, 22.2 mg of desired product (22.2 mg, 0.038 mmol, 100% yield) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 587.3; Rt 0.93 min

Step 3: (S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3-hydroxy-2,2- dimethylpropyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

In a stirring solution of (S)-1-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3- hydroxy-2,2-dimethylpropyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-oxopropan- 2-yl acetate (step 2) (22.2 mg, 0.038 mmol) in methanol (2 mL) was addded K₂C0₃ (26.2 mg, 0.189 mmol). Reaction mixture was stirred at room temperature for 1.5h. Mixture was filtered and submitted to purification by reverse phase chromatography. 8.8 mg of expected product (8.8 mg, 0.013 mmol, 35% yield, 100% pure) were obtained as a TFA salt.

LC/MS (Method B): [M+H]⁺ 545.3; Rt 3.24 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks for the major rotamer are reported): δ 9.09 (1 H, br s), 8.87 (1 H, br s), 7.80-7.87(1 H, m), 7.70-7.76 (1 H, m), 7.30-7.44 (6H, m), 7.1-7.15 (1 H, m), 6.03 (1 H, s), 5.35-5.08 (3H, m), 4.60-4.55 (1 H, m), 4.05-3.85 (2H, m), 3.35-3.20 (2H, m), 3.12-3.04 (2H, m), 2.41-2.31 (1 H, m), 2.01-1.79 (2H, m), 1.35 (3H, d, 6.2Hz), 0.90 (3H, s), 0.80 (3H, s).

Synthesis Example 18

Step 1 : (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-

1 H-imidazol-2-yl)-2,2-dimethyl-4-oxobutyl)propanamido)methyl)-4-fluoropyrrolidine-

1 -carboxylate

Under N₂:To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -( 1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (200 mg, 0.285 mmol) in dry DCM (12 ml) was added Dess-Martin Periodinane (161 mg, 0.380 mmol). The reaction mixture was stirred for 35 min at room temperature. Solution was diluted in DCM, washed with water, brine, dried on Na2SC>4, filtered and concentrated.

No further purification, 199 mg of the desired product (199 mg, 0.285 mmol, 100% yield) were obtained and used in the next step.

LC/MS (Method A): [M+H]⁺ 699.5; Rt1.39 min

Step 2: (3R,4R)-tert-butyl 3-(((2S)-2-acetoxy-N-((1 R,E)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-((tert-butylsulfinyl)imino)-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-2,2-dimethyl-4-oxobutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (250 mg, 0.358 mmol), 2-methylpropane-2-sulfinamide (step 1 ) (217 mg, 1.789 mmol) and CuS04.5H20 (447 mg, 1.789 mmol) were stirred in DCM ( 10 mL) at 50^°C for 42h. The reaction mixture was filtered and precipitate was washed with further DCM. The filtrate (= product) was condensed in vacuo.

No further purification, 496 mg of desired product ( 496 mg, 0.353 mmol, 99% yield, 57% pure) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 802.5; Rt 1.48 min.

Step 3: (3R,4R)-tert-butyl 3-(((2S)-2-acetoxy-N-((1 R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-(1,1-dimethylethylsulfinamido)-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

(3R,4R)-tert-butyl 3-(((2S)-2-acetoxy-N-((1 R,E)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-4-((tert-butylsulfinyl)imino)-2,2 iime

fluoropyrrolidine-1-carboxylate (step 2) (496 mg, 0.353 mmol) was dissolved in MeOH (15 mL). NaBH₄ (66.7 mg, 1.763 mmol), was added slowly. Reaction mixture changed of colour (brown) and bubbled. It was stirred at room temperature for 4h. Residue was diluted in EA, washed with water, then with brine. Organic layer was dried on Na2S04 filtered and concentrated.

No further purification, 379 mg of desired product (379 mg, 0.420 mmol, 1 19% yield (not dry), 89% pure) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 804.7; Rt 1.42 min.

Step 4: (3R,4R)-tert-butyl-3-(((S)-2-acetoxy-N-((R)-4-amino-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

(3R,4R)-tert-butyl 3-(((2S)-2-acetoxy-N-((1 R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-4-(1 ,1-dimethylethylsulfinamido)-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (step 3) (106 mg, 0.1 17 mmol)was dissolved in MeOH (3 mL) at 0^°C under N₂. HCI 4M in dioxane (0.059 mL, 0.235 mmol), was added . Reaction mixture was stirred at 0^°C for 1 h20, then quenched with saturated NaHCC>3 at 0^°C.The solution was diluted in EA, washed with water, then with brine. Organic layer was dried on Na2S04 filtered and concentrated.

No further purification, 82 mg of desired product ( 82 mg, 0.076 mmol, 65% yield, 65% pure) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 700.5; Rt 1.04 min.

Step 5: (3R,4R)-tert-butyl 3-(((S)-N-((R)-4-acetamido-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbutyl)-2- acetoxypropanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

(3R,4R)-tert-butyl-3-(((S)-2-acetoxy-N-((R)-4-amino-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

(step 4) (44 mg, 0.041 mmol) and DIPEA (0.021 mL, 0.123 mmol) in DCM (1 mL). Acetyl chloride (4.35 μΙ, 0.061 mmol) was added. Mixture was stirred at room temperature for 1 h20. Reaction mixture was diluted in DCM.washed successively with water, saturated NaHC03 and brine, dried on Na2S04, filtered and concentrated.

No further purification, 30.3 mg of desired product ( 30.3 mg, 0.041 mmol, 100% yield) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 742.5; Rt 1.25 min. Step 6: (S)-1-(((R)-4-acetamido-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate

(3R,4R)-tert-butyl 3-(((S)-N-((R)-4-acetamido-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-2,2-dimethylbutyl)-2-acetoxypropanamido)methyl)-4-fluoropyrro^ carboxylate

(step 5) (30.3 mg, 0.041 mmol) was dissolved in Acetonitrile ( 2 ml) and water (1 ml). TFA (0.157 ml, 2.042 mmol) was added and the reaction mixture was stirred at 60^°C for 1 h40.

Solution was diluted in EA, washed with saturated NaHCC>3, then with brine. Organic layer was dried on Na2SC>4, filtered and concentrated. No further purification, 30.3 mg of desired product ( 26.2 mg, 0.041 mmol, 100% yield) were obtained and used to the next step.

LC/MS (Method A): [M+H]⁺ 642.4; Rt 0.84 min.

Step 7: (S)-N-((R)-4-acetamido-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-

2,2-dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamide

In a stirring solution of (S)-1-(((R)-4-acetamido-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-2,2-dimethylbutyl)(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)ami

oxopropan-2-yl acetate

(step 6) (26.2 mg, 0.041 mmol) in Methanol (2 mL) was addded K₂C0₃ (28.2 mg, 0.204 mmol) and the reaction mixture was stirred at room temperature for 1 h.

Solution was filtered and submitted to purification by reverse phase chromatography. 7.7 mg of expected product (7.7 mg, 0.01 1 mmol, 26% yield, 100% pure) were obtained as a TFA salt.

LC/MS (Method B): [M+H]⁺ 600.4; Rt 3.09 min. ¹H-NMR (DMSO, 600 MHz): δ 9.06 (1 H, br s), 8.78 (1 H, br s), 7.93 (1 H, d, 3.7Hz), 7.80-7.74 (1 H, m), 7.60-7.55 (1 H, m), 7.44-7.30 (6H, m), 7.17-7.09 (1 H, m), 5.84 (1 H, s), 5.40-5.06 (3H, m), 4.62-4.56 (1 H, m), 4.05-3.90 (2H, m), 3.35-3.21 (2H, m), 2.90-2.80 (2H, m), 2.45-2.35 (1 H, m), 2.00-1.85 (2H, m), 1.73 (3H, s), 1.38-1.41 (1 H, m), 1.35 (3H, d, 6.2Hz),1.18-1.1 1 (2H, m), 0.93 (3H, s), 0.80 (3H, s)

Synthesis Example 19

1 ) HCI, AcOH

2) Pd/C, H₂, MeOH

3 Boc₂0

Step 1 : (R)-2-(((S)-2-hydroxy-1-phenylethyl)amino)-2-(4-methyltetrahydro-2H-pyran- 4-yl)acetonitrile

Solution of 4-methyltetrahydro-2H-pyran-4-carbaldehyde (4.8 g, 37.5 mmol) in DCM (Volume: 50 ml) was cooled to 0 C. (S)-(+)-Phenylglycinol (5.65 g, 41.2 mmol) was added and reaction mixture was stirred at 0 C for 1 h. Tnmethylsilyl cyanide (7.49 ml, 56.2 mmol) was added dropwise and reaction mixture was stirred at room temperature for 24 h. Reaction mixture was quenched with NaOH (2M), extracted with DCM. Organics washed again with NaOH (2 M), dried with Na₂S0₄, filtered and concentrated. Redissolved in THF (20 mL) and acidified with 10 mL HCI (cone). Then basified with NaOH (2M) and extracted with EA. Organics dried with Na₂S0₄, filtered, concentrated and absorbed onto Isolute. The residue was purified by flash chromatography (120 g, silica gel) eluting with heptane/ethyl acetate to yield two diastereomers: desired diastereomer 4.17 g (41 % yield) as a colorless oil and 1.69 g (15% yield) of the undesired diastereomer.

Analytical data for the desired diastereomer: LC/MS (Method A): [M+H]⁺ 275.4, Rt 0.88 min. ¹ H-NMR (DMSO, 400 MHz): δ 7.40-7.25 (5H, m), 5.18 (1 H, t, J = 5.7 Hz), 3.90-3.82 (1 H, m), 3.60-3.33 (6H, m), 3.09 (1 H, d, J = 12.9 Hz), 2.63 (1 H, d, J = 12.9), 1 .68-1 .36 (3H, m), 1.31 -1 .22 (1 H, m), 1 .10 (3H, s).

Analytical data for the second diastereomer:

LC/MS (Method A): [M+H]⁺ 275.4, Rt 0.82 min. ¹ H-NMR (DMSO, 400 MHz): δ 7.42-7.21 (5H, m), 4.79 (1 H, t, J = 5.4 Hz), 3.81 -3.62 (4H, m), 3.55-3.42 (4H, m), 2.69 (1 H, dd, J = 9.5, 4.7 Hz), 1 .71 -1 .55 (2H, m), 1 .48-1 .36 (1 H, m), 1 .34-1 .22 (1 H, m), 1 .09 (3H, s).

(R)-2-((tert-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid

Step 2: (R)-2-(((S)-2-hydroxy-1 -phenylethyl)amino)-2-(4-methyltetrahydro-2H-pyran-4- yl)acetonitrile (step 1 ) (4.17 g, 15.20 mmol) was heated in a mixture of HCI cone (57.7 ml, 608 mmol, 32%) and AcOH (25 ml, 437 mmol) at 85^°C for 3h. Concentrated, coevaporated with toluene (twice). Used in the next step without any purification.

Step 3: Residue was dissolved in MeOH (Volume: 70 mL), reaction mixture was purged with N₂, then Pd-C (1 .608 g, 1 .51 1 mmol, 10%) was added and a baloon filled with hydrogen was attached to septa. Reaction mixture was purged with hydrogen, then stirred under hydrogen at room temperature for 3 days until starting material was consumed. Filtered through celite, eluting with MeOH and concentrated.

Step 4: Residue was suspended in DCM (Volume: 40 mL), diisopropylamine (6.50 mL, 30.2 mmol) followed by Boc₂0 (3.31 g, 15.2 mmol) were added and reaction mixture was stirred at RT for 16 h. Concentrated, diluted with NaOH (0.2 M) and washed with DCM. Aqueous layer acidified to pH 1 with HCI (4 M) and product extracted with EA. Organics dried with Na₂S0₄, filtered and concentrated to give desired product (1 .89 g, 6.57 mmol, 44 % yield). ¹H-NMR (DMSO, 400 MHz): δ 12.59 (1 H, s), 6.92 (1 H, d, J = 9.2 Hz), 3.98 (1 H, d, J = 9.2 Hz), 3.69-3.55 (2H, m), 3.54-3.42 (2H, m), 1.70-1.45 (2H, m), 1.39 (9H, s), 1.36-1.21 (2H, m), 0.99 (3H, s).

(R)-2-(2,5-difluorophenyl)-2-oxoethyl-2-((tert-butoxycarbonyl)amino)-2-(4- methyltetrahydro-2H-pyran-4-yl)acetate

The title compound was prepared by a method similar to that described in Scheme 1 by replacing (R)-2-((tert-butoxycarbonyl)amino)-2-(tetrahydro-2H-pyran-4-yl)acetic acid with (R)-2-((tert-butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetic acid); Colorless solid (6.9 g, 16.14 mmol, 88% yield).

LC/MS (Method A): [M+H]⁺ 428.2, Rt 1.16 min.

(R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro-2H- pyran-4-yl)methyl)carbamate

The title compound was prepared by a method similar to that described in Scheme 1 by replacing (R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-2- (tetrahydro-2H-pyran-4-yl)acetate with (R)-2-(2,5-difluorophenyl)-2-oxoethyl-2-((tert- butoxycarbonyl)amino)-2-(4-methyltetrahydro-2H-pyran-4-yl)acetate; Yellow foam (6.58 g, 16.14 mmol, 100% yield).

LC/MS (Method A): [M+H]⁺ 408.2, Rt 1.06 min. (R)-tert-butyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro- 2H-pyran-4-yl)methyl)carbamate

The title compound was prepared by a method similar to that described in Scheme 1 by replacing (R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)carbamate with (R)-tert-butyl ((4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methyl)carbamate; Yellowish solid (6.58 g, 10.58 mmol, 78% yield). LC/MS (Method A): [M+H]⁺ 498.3, Rt 1.44 min.

(R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro-2H-pyran- 4-yl)methanamine

The title compound was prepared by a method similar to that described in Scheme 1 by replacing (R)-tert-butyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)carbamate with (R)-tert-butyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(4-methyltetrahydro-2H-pyran-4-yl)methyl)carbamate; Yellowish solid (4.23 g, 10.58 mmol, 99% yield, as a TFA salt).

LC/MS (Method A): [M+H]⁺ 398.2, Rt 0.90 min. (3R,4R)-benzyl 3-((((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1- carboxylate

The title compound was prepared by a method similar to that described in Scheme 1 by replacing (R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methanamine with (R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methanamine; Colorless oil (360 mg, 0.55 mmol, 44% yield). LC/MS (Method B): [M+H]⁺ 633.3, Rt 6.05 min.

(3R,4R)-benzyl 3-((1 -((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methyl)-3-((S)-1 -hydroxypropan-2- yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate

The title compound was prepared by a method similar to that described in General Procedure for Synthesis of Urea Compounds.

Colorless oil (207 mg, 0.24 mmol, 42% yield, 85% pure).

LC/MS (Method A): [M+H]⁺ 734.3, Rt 1.32 min. 1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro-2H- pyran-4-yl)methyl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-3-((S)-1- h yd roxy pro pa n -2-y I ) u rea

The title compound was prepared by a method similar to that described in General Procedure for Cbz Deprotection.

TFA salt was converted to a free base by passing through a PL-HC0₃ cartridge. Colorless solid (8 mg, 0.012 mmol, 14% yield).

LC/MS (Method B): [M+H]⁺ 600.2, Rt 3.71 min. ¹H-NMR (DMSO, 600 MHz) (mixture of rotamers): δ 7.87-7.80 (1 H, m), 7.77-7.68 (1 H, m), 7.43-7.26 (6H, m), 7.13-7.06 (1 H, m), 6.13-6.05 (1 H, m), 5.67-5.62 (1 H, m), 5.37-5.22 (2H, m), 5.94-4.91 (1 H, m), 4.66 (1 H, br s), 3.95-3.75 (2H, m), 3.70-3.55 (2H, m), 3.46-3.38 (2H, m), 3.27-3.17 (2H, m), 2.97-2.81 (1 H, m), 2.68-2.54 (1 H, m), 2.08-2.02 (1 H, m), 1.89-1.74 (1 H, m), 1.56-1.44 (2H, m), 1.35- 1.28 (2H, m), 1.17-0.98 (7H, m). Some signals hidden under the solvent peak.

Synthesis Example 20

Step 1 : (S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)morpholine-2-carboxylic acid

(S)-morpholine-2-carboxylic acid hydrochloride [CAS 154731-81-4] (100 mg, 0.597 mmol) and SODIUM BICARBONATE (251 mg, 2.98 mmol) were dissolved in H₂0 (3 ml) and a solution of Fmoc-OSu [Aldrich, 8291 1-69-1] (302 mg, 0.895 mmol) in Dioxane (4 ml) was added. RM was stirred at RT for 3 h. Partitioned between EtOAc and NaHC0₃ (sat.). Aqueous layer washed with EA and then acidified to pH3 (with HCI 1 M) and extracted with. Organics dried with Na2S04, filtered and concentrated. 210 mg of the desired product were obtained as a colorless oil.

Colorless oil (210 mg, 0.582 mmol, 98% yield, 98% pure). LC/MS (Method A): [M+H]⁺ 354.1 , [M+NH₄]⁺ 371.1 , Rt 0.93 min.

Step 2: (S)-(9H-f luoren-9-yl)methyl 2-(((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)carbamoyl)morpholine-4-carboxylate

(S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)morpholine-2-carboxylic acid (Step 1 ) (210 mg,

0.582 mmol) was dissolved in DCM (10 ml), then DMF (7.95 μΙ, 0.103 mmol) were added, followed by OXALYL CHLORIDE (0.081 ml, 0.924 mmol). Reaction mixture was stirred at room temperature for 1 h, concentrated and coevaporated twice with toluene. Residue was dissolved in 10 mL of DCM and cooled to 0^°C, pyridine (0.332 ml, 4.10 mmol) followed by DMAP (2.507 mg, 0.021 mmol) were added. (3R,4R)-tert-butyl 3-((((R)-(1- benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate (120 mg, 0.205 mmol) was finally added dropwise as a solution in 5 ml_ of DCM. RM was stirred at 0^°C for 15 min, then at room temperature for 2 h. Diluted with NaHC0₃ (sat.) and extracted with DCM, dried with Na₂S0₄, filtered and absorbed onto Isolute. Residue purified by flash chromatography (40g, silica gel) eluting with heptane/ethyl acetate to afford 135 mg of the desired product as a colorless oil.

Colorless oil (135 mg, 0.142 mmol, 69% yield, 97% pure).

LC/MS (Method A): [M+H]⁺ 920.4 Rt 1.53 min.

Step 3: (S)-N-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2- carboxamide

Fmoc group was removed by reacting with piperidine in DMF.

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 16 h) after purification by reverse phase column chromatography (23 mg, 0.028 mmol, 65% yield, as a double TFA salt).

LC/MS (Method B): [M+H]⁺ 598.2, Rt 2.23 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, 120^°C): δ 7.83-7.77 (1 H, m), 7.68 (1 H, br s), 7.45-7.35 (3H, m), 7.30-7.22 (3H, m), 7.12-7.05 (1 H, m), 5.42-5.13 (4H, m), 4.71 (1 H, br s), 4.05-3.92 (2H, m), 3.90-3.80 (2H, m), 3.73-3.64 (2H, m), 3.50-3.15 (9H, m), 2.72-2.61 (2H, m), 2.30-2.10 (1 H, m), 1.50- 1.20 (3H, m), 0.97-0.80 (1 H, m).

Step 1 : (R)-(9H-f luoren-9-yl)methyl 2-(((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)carbamoyl)morpholine-4-carboxylate

The title compound was prepared by a method similar to that described for the (S)- diastereomer. Purified by flash chromatography (40g, silica gel) eluting with heptane/ethyl acetate to afford 104 mg of the desired product as a colorless oil.

Colorless oil (135 mg, 0.1 13 mmol, 34% yield).

LC/MS (Method A): [M+H]⁺ 920.3 Rt 1.54 min.

(S)-diasteromer was obtained as well: colorless oil (190 mg, 0.207 mmol, 62% yield). LC/MS (Method A): [M+H]⁺ 920.3 Rt 1.56 min. Step 2: (R)-N-((R)-(1-benzvl-4-(2.5-difluorophenvl)-1 H-imidazol-2-vl)(tetrahvdro-2H- pyran-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2- carboxamide

Fmoc group was removed by reacting with piperidine in DMF.

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 16 h) after purification by reverse phase column chromatography (15 mg, 0.018 mmol, 98% yield, as a double TFA salt). LC/MS (Method B): [M+H]⁺ 598.2, Rt 2.55 min.

Synthesis Example 21. 4-hydroxy-THP-Core Scaffold Synthesis

4-((2R,5S)-5-isopropyl-3,6-dimethoxy-2,5-dihydro

Over a pre-cooled (-78 C) solution of (S)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (5.4 mL, 30 mmol, 1 eq) in TH F (60 mL), nBuLi (1 .6 M in Hexane, 31 .5 mmol, 1 .1 eq) was added. The reaction mixture was stirred at -78 C for 1 h. After 1 h, dihydro-2H-pyran- 4(3H)-one (3 g, 30 mmol, 1 eq) was added as a solution in THF (40 mL). The reaction mixture was stirred at -20 °C. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was quenched with a solution of AcOH (1 .8 mL, 31 .5 mmol, 1 .1 eq) in THF ( 15 mL), and allowed to warm to rt. The reaction mixture was partioned between Et₂0 and H₂0. The organic layer was separated, dried over Na2SC>4, filtered, and solvent evaporated under reduce pressure. Crude was purified by normal phase column chromatography (PrepLC Method B) to give the desire prodcut (7.3 g, 25.7 mmol, 86 %). LC/MS (uplc): MH+ 285.2, 0.96 min (Method A).

(R)-methyl 2-amino-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetate.

Over a solution of 4-((2R,5S)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2- yl)tetrahydro-2H-pyran-4-ol (4.3g, 15.1 mmol, 1 eq) in THF ( 107 mL), was added HCI (0.2 Nm 151 mL, 30.2 mmol, 2 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was quenched with NaOH (1 .0M) until pH=8, and used as a solution in the next step. LC/MS (uplc): MH+190.1 , 0. 17 min (Method D, Polar Method).

(R)-methyl 2-(((benzyloxy)carbonyl)amino)-2-(4-hydroxytetrahydro-2H-pyran-4-yl)acetate.

Over the solution from the previous step (6.62g, 14 mmol, 1 eq), were added NaHC0₃ (4.1 g, 49 mmol, 3.5 eq) and Cbz-CI (5 mL, 35 mmol, 2.5 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was partioned between EtOAc (100 mL) and H₂0 (100 mL). Organic layer was separated, dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. Crude was purified by normal phase column chromatography (PrepLC Method A) to give the desire product (5.4 g, 16.7 mmol, 60 %). LC/MS (uplc): MH+ 324.2, 0.78 min.

(R)-methyl 2-(((benzyloxy)carbonyl)amino)-2-(4-((tert-butyldimethylsilyl)oxy)tetrahydro- 2H-pyran-4-yl)acetate.

Over an ice-cooled solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-2-(4- hydroxytetrahydro-2H-pyran-4-yl)acetate (2.3 g, 6.4 mmol, 1 eq) in CH₂CI₂ (5 mL), were added 2,6-lutidine (5 mL, 42.9 mmol, 6.7 eq), and TBSOTf (5 mL, 21.8, 3.4 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was partioned between H₂0 (50 mL) and CH₂CI₂ (50 mL). The organic layer was separated, dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. Crude was purified by normal phase chromatography (PrepLC Method A) to give the desire product (2.1 g, 4.6 mmol, 73 %). LC/MS (uplc): MH+ 438.2, 1.42 min (Method A). (R)-2-(((benzyloxy)carbonyl)amino)-2-(4-((tert-butyldimethylsilyl)oxy)tetrah

4-yl)acetic acid.

Over a solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-2-(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)acetate (2.1 g, 4.9 mmol, 1 eq) in THF, was added KOH (1 .0 M, 14.7 ml_, 14.7 mmol). The reaction mixture was stirred at room temperature. Upon completion of the reaction (uplc, Method A), the reaction mixture was quenched with HCI (1 .0 M) until pH=4. The reaction was partioned between EtOAc (60 ml_), and H₂0 (60 ml_). The organic layer was separated, dried over Na₂S0₄, filtered and solvent evaporated under reduce pressure to give the desire product (2.2g, 4.8 mmol, 99%). Crude was used without any further purification. LC/MS (uplc): MH+424.3, 1 .25 min (Method A).

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-(((benzyloxy)carbonyl)amino)-2-(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)acetate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 578.3, 1 .49 min (Method A).

(S)-benzyl ((4-((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)(4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)methyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 558.3, 1 .48 min (Method A).

(S)-benzyl ((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)methyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase column

chromatography (PrepLC Method A). LC/MS (uplc): MH+ 648.6.3, 1 .64 min (Method A).

(S)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)methanamine.

For the preparation of this compound, follow procedure described for general Cbz- deprotection. Crude used without any further purification. LC/MS (uplc): MH+ 514.6, 1.40 min (Method A).

(3R,4R)-tert-butyl 3-((((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-fluoropyrrolidine-

1-carboxylate.

chromatography (PrepLC Method A). LC/MS (uplc): MH+ 715.6, 1.65 min (Method A).

(3R,4R)-tert-butyl 3-((1-((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-((tert- butyldimethylsilyl)oxy)tetrahydro-2H-pyran-4-yl)methyl)-3-((S)-1-hydroxypropan-2- yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate.

chromatography (PrepLC Method A). LC/MS (uplc): MH+ 817.5, 1.59 min (Method A). Synthesis Example 22. Cvclopropanol-Core Scaffold Synthesis

Starting from (R)-2-(((benzyloxy)carbonyl)amino)-2-(1-hydroxycyclopropyl)acetic acid prepared following described protocol: Esposito, A.; Paolo-Piras, P.; Ramazzotti, D.; Taddei, M. Org. Lett. 2001 , 3, 3273-3275.

(R)-2-(((benzyloxy)carbonyl)amino)-2-(1-((tert-butyldimethylsilyl)oxy)cyclopropyl)acetic acid.

Over a solution of (R)-2-(((benzyloxy)carbonyl)amino)-2-(1-hydroxycyclopropyl)acetic acid

(3g, 1 1.3 mmol, 1 eq) and Imidazole (2.3 g, 34 mmol, 3 eq) in DMF (23 mL), was added

TBSCI (4.3 g, 28.3 mmol, 2.5 eq). The reaction mixture was stirred at room temperature.

Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was partioned between EtOAc (100 mL) and HCI (1.0 M, 100 mL). The organic layer was separated, dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure to give a yellow oil. Crude used without any further purification. LC/MS (uplc): MH+ 380.3, 1.25 min (Method A).

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-(((benzyloxy)carbonyl)amino)-2-(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)acetate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 534.4, 1.51 min (Method A).

(S)-benzyl ((1-((tert-butyldimethylsilyl)oxy)cyclopropyl)(4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)methyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 514.8, 1.47 min (Method A).

(S)-benzyl ((1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)methyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): MH+ 604.1 , 1.64 min (Method A).

(S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)methanamine.

For the preparation of this compound, follow procedure described for general Cbz- deprotection. Crude used without any further purification. LC/MS (uplc): MH+ 470.3, 1.26 min (Method A).

(3R,4R)-benzyl 3-((((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): MH+ 706.4, 1.62 min (Method A).

(3R,4R)-benzyl 3-((1-((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4- fluoropyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): MH+ 807.6, 1.60 min (Method A).

(3R,4R)-tert-butyl 3-((1-((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((tert- butyldimethylsilyl)oxy)cyclopropyl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4- fluoropyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure for protecting group exchange (Cbz-deprotection, Boc-protection) described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method B). Cbz-deprotection: LC/MS (uplc): M+ 672.4, 1.25 min (Method A). Boc-protection: LC/MS (uplc): M+ 772.7. 1.61 min (Method A).

Synthesis Example 23. 2-Methoxypropyl-Core Scaffold Synthesis

(R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-methylbutanoic acid.

To an ice-cooled solution of NaH (60% mineral oil, 2.5 g, 64. mmol, 3 eq), and (R)-2- ((tert-butoxycarbonyl)amino)-3-hydroxy-3-methylbutanoic acid (5 g, 21.4 mmol, 1 eq) in THF, Mel (1.6 mL, 25.7 mmol, 1.2 eq) was added and the reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction was quenched with HCI (1.0 M) until pH=3. The crude reaction was partioned between EtOAc (100 mL) and HCI (1.0 M, 100 mL), the two layers were separated, and the organic layer was washed with HCI (1.0 M, 3 x 100 mL), dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure to give (R)-2-((tert-butoxycarbonyl)amino)- 3-methoxy-3-methylbutanoic acid. The crude was used without any further purification in the next step. LC/MS (uplc): MH+ 248.2, 0.79 mi (Method D, Polar Method).

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-3-methoxy-3- methylbutanoate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 402.2, 1.20 min (Method A).

(S)-tert-butyl (1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 382.3, 1.08 min (Method A).

(S)-tert-butyl (1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 472.4, 1.44 min (Method A).

(S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2-methylpropan-1- amine.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 372.4, 0.88 min (Method A).

(3R,4R)-tert-butyl 3-((((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy- 2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 573.7, 1.38 min (Method A).

(3R,4R)-tert-butyl 3-((1-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2- methoxy-2-methylpropyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-

1-carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 674.4, 1.34 min (Method A).

(3R,4S)-tert-butyl 3-((((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy- 2-methylpropyl)amino)methyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate.

To a solution of (S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropan-1 -amine (0.7 g, 0.72 mmol) and sodium triacetoxyborohydride (8.1 g, 38 mmol) in CH₂CI₂ (3 mL) was added (3S,4R)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4- formylpyrrolidine-1-carboxylate in CH₂CI₂ (1 mL). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was partioned between CH₂CI₂, and H₂0. The organic layer was separated and washed with sat. solution NaHC0₃ (twice), and H₂0 (twice), then dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. Crude purified by normal phase column chromatography (PrepLC Method A) to give (3R,4S)-tert-butyl 3-((((S)-1-(1- benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2-methylpropyl)amino)methyl)- 4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate (250 mg, 0.33 mmo, 46%). LC/MS (uplc): MH+ 685.9, 1.76 min (Method A).

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-

2-yl)-2-methoxy-2-methylpropyl)propanamido)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): M+ 799.5, 1.69 min (Method A).

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-2-methoxy-2-methylpropyl)propanamido)methyl)-4-hydroxypyrrolidine-1-carboxylate.

Over a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((S)-1-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2-methylpropyl)propanamido)methyl)-4- ((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate (108 mg, 0.13 mmol, 1 eq), in THF (2 ml_), TBAF (1.0 M in THF, 0.15 mmol, 1.1 eq) was added. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction was partioned between NH₄CI

(saturated solution) and CH₂CI₂. The organic layer was separated and washed with NaCI (saturated solution), dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. Crude purified by normal phase column chromatography (PrepLC Method A). LC/MS (uplc): MH+ 685.6, 1.34 min (Method A).

(3R,4S)-tert-butyl 3-(((S)-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2- methoxy-2-methylpropyl)-2-hydroxypropanamido)methyl)-4-hydroxypyrrolidine-1- carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method B). LC/MS (uplc): MH+ 643.3, 1.30 min (Method A).

(S)-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropyl)-2-hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamide.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude purified by normal phase column chromatography (PrepLC Method B). 17.6 mg, 0.031 mmol, 71 %.

¹H-NMR (DMSO, 600 MHz): δ 7.93-7.87 (1 H, m), 7.80-7.71 (1 H, m), 7.45-7.28 (6H, m), 7.15-7.06 (1 H, m), 6.00-5.87 (1 H, m), 5.46-5.31 (1 H, m), 5.18-5.05 (1 H, m), 4.93-4.77 (2H, m), 4.77-4.64 (1 H, m), 3.69-3.53 (3H, m), 2.95-2.83 (3H, m), 2.75-2.63 (1 H, m), 2.39-2.28 (1 H, m), 2.26-2.07 (1 H, m), 1.59-1.45 (1 H, m), 1.44-1.34 (3H, m), 1.33-1.22 (3H, m), 1.21- 1.12 (1 H, m), 0.79-0.69 (3H, m). LC/MS (uplc): MH+ 543.3, 0.91 min (Method A).

Synthesis Example 24. 2-hydroxypropyl-Core Scaffold Synthesis

(R)-2-(2,5-difluorophenyl)-2-oxoethyl 2-((tert-butoxycarbonyl)amino)-3-hydroxy-3- methylbutanoate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 388.3, 1.04 min (Method A).

(S)-tert-butyl (1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-hydroxy-2- methylpropyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH+ 368.5, 0.96 min (Method A).

(S)-tert-butyl (1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-hydroxy-2- methylpropyl)carbamate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): MH₂+ 459.5, 1 .36 min (Method A).

(S)-1 -amino-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methylpropan-2-ol.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude used without any further purification. LC/MS (uplc): MH₂+ 359.3, 0.80 min (Method A).

(3R,4R)-benzyl 3-((((S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-hydroxy-2- methylpropyl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): MH+ 593.3, 1 .24 min (Method A).

(3R,4R)-benzyl 3-((((S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-((tert- butyldimethylsilyl)oxy)-2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate.

Over an ice-cooled solution of (3R,4R)-benzyl 3-((((S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)-2-hydroxy-2-methylpropyl)amino)methyl)-4-fluoropyrrolidine-1 - carboxylate (1 .5 g, 0.94 mmol, 1 eq) in CH₂CI₂ (5 mL), 2,6-lutidine (0.7 mL, 5.7 mmol, 6 eq) and TBSOTf (0.7 mL, 3.8 mL, 4 eq) were added. The reaction was stirred at 0 °C. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction was partioned between H₂0 (20 mL) and CH₂CI₂ (20 mL). The organic layer was separated, dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. Crude was purified by normal phase chromatography (PrepLC Method A) to give the desire product (0.13 mmol, 130 mg, 14 %). LC/MS (uplc): MH+ 707.3, 1 .70 min (Method A).

(3R,4R)-benzyl 3-((1 -((S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-((tert- butyldimethylsilyl)oxy)-2-methylpropyl)-3-((S)-1 -hydroxypropan-2-yl)ureido)methyl)-4- fluoropyrrolidine-1 -carboxylate.

For the preparation of this compound, follow procedure described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A). LC/MS (uplc): M+ 808.4, 1.64 min (Method A).

(3R,4R)-tert-butyl 3-((1-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-((tert- butyldimethylsilyl)oxy)-2-methylpropyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)-4- fluoropyrrolidine-1-carboxylate.

For the preparation of this compound, follow procedure for protecting group exchange (Cbz-deprotection, Boc-protection) described for analogous compound of the THP series. Crude was purified by normal phase chromatography (PrepLC Method A).

Cbz-deprotection: LC/MS (uplc): M+ 674.4, 1.33 min (Method A).

Boc-protection: LC/MS (uplc): M+ 774.4. 1.66 min (Method A).

Synthesis example 25. (3S,4R)-benzyl 3-((tert-butyldimethylsilyl)oxy)-4-vinylpyrrolidine- 1-carboxylate

(3S,4R)-benzyl 3-hydroxy-4-vinylpyrrolidine-1-carboxylate (4.00 g, 16.18 mmol) in DCM (54 ml) was treated with imidazole (1.65 g, 24.3 mmol) followed by tert-butyldimethylsilyl chloride (2.93 g, 19.4 mmol) and the reaction mixture was stirred at room temperature for 16 hours. The mixture was quenched with saturated aqueous NH₄CI solution and the organic layer was extracted with DCM. The organic layers were combined dried over Na₂S0₄, filtered and concentrated to dryness to afforded the title compound as an pale yellow oil in 102% (unpurified) yield; UPLC-MS: Rt = 1.56mins; MS m/z [M+H]⁺ 362.2; Method A.

(3S,4S)-benzyl 3-((tert-butyldimethylsilyl)oxy)-4-((R)-1 ,2-dihydroxyethyl)pyrrolidine-1- carboxylate

To a solution of (3S,4R)-benzyl 3-((tert-butyldimethylsilyl)oxy)-4-vinylpyrrolidine-1- carboxylate (4.90 g, 13.6 mmol) in a mixture of tert butanol (40 ml) and water (28 ml) at 0 °C was added slowly a solution of potassium permanganate (1.63 g, 10.3 mmol) and sodium hydroxide (0.352 g, 8.81 mmol) in water (10 ml). The reaction mixture was stirred at 0 °C for 1 hour. The mixture was extracted with DCM. The organic layers were combined dried over Na₂S0₄, filtered through celite and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as a pale yellow oil in 64% yield; UPLC-MS: Rt = 1.21 mins; MS m/z [M+H]⁺ 396.1 ; Method A.

(3S,4S)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-((R)-1 ,2-dihydroxyethyl)pyrrolidine-1 - carboxylate

a solution of (3S,4S)-benzyl 3-((tert-butyldimethylsilyl)oxy)-4-((R)-1 ,2- Dxyethyl)pyrrolidine-1 -carboxylate (3.41 g, 8.62 mmol) in methanol (55 ml) under argon was added 10% palladium on carbon (0.917 g, 0.862 mmol) followed by ammonium formate (6.52 g, 103 mmol) and the reaction mixture was stirred at 50 °C for 30 mins. The mixture was then cooled to RT, filtered over celite and washed with methanol. To the filtrate was added di-tert-butyl dicarbonate (2.63 g, 12.1 mmol) and the reaction mixture was stirred at RT for 1 h. The methanol was removed by evaporation and the reaction mixture was then extracted with ethyl acetate and a saturated aqueous NaHC0₃ solution. The organic layer was then washed with brine and the organic phase were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% (10% methanol in DCM) in heptane afforded the title compound as an pale yellow oil in 85% yield; UPLC- MS: Rt = 1.25 mins; MS m/z [M+H]⁺ 362.1 ; Method A.

(3S,4R)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-formylpyrrolidine-1-carboxylate

To a solution of (3S,4S)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-((R)-1 ,2- dihydroxyethyl) pyrrolidine-1-carboxylate (2.95 g, 8.16 mmol) in a mixture of methanol (44 ml) and water (1 1 ml) at 0 °C was added sodium periodate (2.09 g, 9.79 mmol) and the reaction mixture was stirred at RT for 45 mins. The reaction mixture was filtered and the methanol was removed by evaporation. Water (15 ml) was added and the reaction was extracted with DCM. The organic layers were combined dried over Na₂S0₄, filtered through celite and concentrated to dryness to afford the title compound as a pale yellow oil in 100% yield; ¹H NMR (400 MHz, CDCI3) δ 9.69 (1 H, br s), 4.57-4.53 (1 H, m), 3.71- 3.49 (3H, m), 3.24-3.19 (1 H, m), 3.01-2.95 (1 H, m), 1.45 (9H, s), 0.88 (9H, s), 0.08 (6H, s).

(3R,4S)-tert-butyl 3-((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-

2H-pyran-4-yl)methyl)amino)methyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1- carboxylate

To a solution of sodium triacetoxyborohydride (5.77 g, 27.2 mmol) in DCM (14 ml) was added (R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methanamine (2.09 g, 5.44 mmol). To the reaction mixture was added slowly a solution of (3S,4R)-tert-butyl 3-((tert-butyldimethylsilyl)oxy)-4-formylpyrrolidine-1 - carboxylate (2.69 g, 8.16 mmol) in DCM (14 ml) and the reaction mixture was stirred at RT for 16h. The mixture was then quenched with the addition of water and the reaction mixture was extracted with a 1 M aqueous solution of Na₂S₂0₃ and DCM. The organic layer was washed with a saturated aqueous solution of NaHC0₃ then brine. The organic extractes were combined, dried over Na2SC>4, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 95% yield; UPLC-MS: Rt = 1 .64 mins; MS m/z [M+H]⁺ 697.8; Method A.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-((((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate (1.79 g, 2.57 mmol) in dry DCM (26 ml) was slowly added at 0°C N-ethyl-N-isopropylpropan-2-amine (0.63 ml, 3.60 mmol) followed by (S)-1-chloro-1-oxopropan-2-yl acetate (0.36 ml, 2.83 mmol). The reaction mixture was stirred at 0 °C for 5 mins then allowed to warm to RT and was stired for an additional 1.5h. The reaction was quenched with a saturated aqueous solution of NaHC0₃ and extracted with DCM. The organic extractes were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 88% yield; UPLC-MS: Rt = 1.63 mins; MS m/z [M+H]⁺ 81 1.2; Method A.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-hydroxypyrrolidine-1- carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)- 4-((tert-butyldimethylsilyl)oxy) pyrrolidine-1-carboxylate (1.92 g, 2.37 mmol) in THF (12 ml) was added slowly tetrabutylammonium fluoride (0.619 g, 2.37 mmol). The reaction mixture was stirred at RT for 30 mins. The reaction was quenched with a saturated aqueous solution of NH₄CI and extracted with DCM. The organic extractes were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 66% yield; UPLC-MS: Rt = 1.25 mins; MS m/z [M+H]⁺ 696.9; Method A..

(3S,4R)-tert-but l 3-((((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)amino)methyl)-4-((tert-butyldimethylsilyl)oxy)pyrroli^

carboxylate

The product was synthesized in an analogous way as example 25 using (3R,4S)-benzyl 3-hydroxy-4-vinylpyrrolidine-1 -carboxylate instead; 105% yield; UPLC-MS: Rt = 1 .63 mins; MS m/z [M+H]⁺ 697.0; Method A.

(3S,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1 -carboxylate

quantitative yield; UPLC-MS: Rt = 1.63 mins; MS m/z [M+H]⁺ 81 1.0; Method A.

(3S,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-hydroxypyrrolidine-1- carboxylate

32% yield; UPLC-MS: Rt = 1.26 mins; MS m/z [M+H]⁺ 696.9; Method E.

Synthesis Example 26.

(R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,2-dimethylpropan-1-am

Prepared as described in WO2008086122 A2, 2008; P.50-52.

(3R,4S)-tert-butyl 3-((((R)-1 -(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- )-4-((tert-butyldimethylsilyl)oxy)pyrrolidin

The product was synthesized in an analogous way as example 1 using (R)-1 -(1 -benzyl-4- (2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylpropan-1 -amine;93% yield; UPLC-MS: Rt = 2.66 mins; MS m/z [M+H]⁺ 669.5; Method E.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-

2-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-1 - carboxylate

39% yield; UPLC-MS: Rt = 3.86 mins; MS m/z [M+H]⁺ 783.6; Method E.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-hydroxypyrrolidine-1 -carboxylate

43% yield; UPLC-MS: Rt = 2.96 mins; MS m/z [M+H]⁺ 669.4; Method E. Synthesis Example 27.

(3R,4S)-tert-butyl 3-(( 1 -((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-

2H-pyran-4-yl)methyl)-3-((R)-1 -hydroxypropan-2-yl)ureido)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1 -carboxylate

To a solution of phosgene (20% in toluene, 0.76 mL, 1 .4 mmol) in DCM (7.2 mL) was added a solution of (3R,4S)-tert-butyl 3-((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate (0.50 g, 0.717 mmol) and triethylamine (0.30 mL, 2.15 mmol) in DCM (7.2 mL). The reaction mixture was stirred at room temperature for 45 mins. L-Alaninol (1 .26 mL, 16.1 mmol) was added and the reaction stirred at 40 °C for 16h. The reaction was concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow solid in 76% yield; UPLC-MS: Rt = 1 .54 mins; MS m/z [M+H]⁺ 797.9; Method A.

(3R,4S)-tert-butyl 3-((3-((R)-1 -acetoxypropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-

1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-((tert- butyldimethylsilyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-((1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-((R)-1 -hydroxypropan-2-yl)ureido)methyl)-4- ((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate (0.43 g, 0.539 mmol) in dry DCM (5.4 ml) was slowly added at RT pyridine (0.87 ml, 10.8 mmol) followed by acetic anhydride (1.01 ml, 10.8 mmol). The reaction mixture was stirred at RT for 16h. The reaction was concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow solid in 89% yield; UPLC-MS: Rt = 1.60 mins; MS m/z [M+H]⁺ 840.0; Method A.

(3R,4S)-tert-butyl 3-((3-((R)-1-acetoxypropan-2-yl)-1-((R)-(1-benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-hydroxypyrrolidine-1-

carboxylate

The product was synthesized in an analogous way as in example 1 ; 76% yield; UPLC- MS: Rt = 1.23 mins; MS m/z [M+H]⁺ 725.9; Method A.

Synthesis Example 28.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-

((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-

4-hydroxypyrrolidine-1 -carboxylate (1 .32 g, 1 .89 mmol) in DCM (19 ml) was added at 0 °C methanesulfonyl chloride (0.74 ml, 9.47 mmol) followed by triethylamine (1 .3 ml, 9.47 mmol) and the reaction mixture was stirred at 0 °C for 1 .5h. The reaction was concentrated to dryness. Purification of the crude product by chromatography ono silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as a pale yellow solid in 47% yield; UPLC-MS: Rt = 2.67 mins; MS m/z [M+H]⁺ 775.5; Method E. (3R,4S)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-4-

((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)- 4-((methylsulfonyl)oxy) pyrrolidine-1 -carboxylate (693 mg, 0.894 mmol) in MeOH (9 ml) was added potassium carbonate (148 mg, 1 .07 mmol) and the reaction mixture was stirred at RT for 30 mins. The reaction was diluted with brine and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the crude title compound as a pale yellow solid in quantitative yield and was used in the next step without further purification; UPLC-MS: Rt = 2.54 mins; MS m/z [M+H]⁺ 733.4; Method E.

(3R,4S)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)propanamido)methyl)-

4-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-4- ((methylsulfonyl)oxy) pyrrolidine-1-carboxylate (655 mg, 0.894 mmol) in DCM (8.9 ml) was added 1 H-imidazole (0.91 mg, 1.34 mmol) followed by tert-butylchlorodimethylsilane (202 mg, 1.34 mmol) and DMAP (1 mg, cat) and the reaction mixture was stirred at RT for 4h. Additional tert-butylchlorodimethylsilane (229 mg, 1.52 mmol) was added followed by 1 H-imidazole (0.55 mg, 0.804 mmol) and the reaction was stirred for another 2 days. The reaction was diluted with brine and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as a offwhite solid in 36% yield; UPLC-MS: Rt = 3.32 mins; MS m/z [M+H]⁺ 847.6; Method E.

(3R,4R)-tert-butyl 3-acetoxy-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy)propanamido)methyl)pyrrolidine-1-carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy)propanamido)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1- carboxylate (52 mg, 0.061 mmol) in DMF (0.6 ml) was added potassium acetate (12 mg, 0.123 mmol) and the reaction mixture was stirred at 1 10 °C for 2h. The reaction was cooled to RT and diluted with water and extracted with MTBE. The organic extracts were combined, dried over Na₂SC>4, filtered and concentrated to dryness to give the crude product as a yellow solid in 88% yield which was used in the next step without further purification; UPLC-MS: Rt = 3.41 mins; MS m/z [M+H]⁺ 81 1.6; Method E. (3R,4R)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)propanamido)m

4-hydroxypyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-acetoxy-4-(((S)-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy) propanamido)methyl) pyrrolidine-1 -carboxylate (44 mg, 0.054 mmol) in MeOH (0.55 ml) was added potassium carbonate (8 mg, 0.054 mmol) and the reaction mixture was stirred at RT for 20 mins. The reaction was concentrated to dryness to afforded the title compound as a pale yellow solid in quantitative yield and was used in the next step without further purification; UPLC-MS: Rt = 3.17 mins; MS m/z [M+H]⁺ 769.6; Method E.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)propanamido)methyl)-

4-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophi imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy)propanamido)m (160 mg,

0.208 mmol) in DCM (2.1 ml) was added at 0 °C methanesulfonyl chloride (0.08 ml, 1.04 mmol) and triethylamine (0.15 ml, 1.04 mmol) and the reaction mixture was stirred at 0 °C for 1.5 h. The reaction mixture was concentrated to dryness and purification of the crude product was performed by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as a pale yellow solid in 85% yield; UPLC-MS: Rt = 3.27 mins; MS m/z [M+H]⁺ 847.6; Method E.

(3S,4R)-tert-butyl 3-azido-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy)propanamido)methyl)pyrrolidine-1-carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert- butyldimethylsilyl)oxy)propanamido)methyl)-4-((methylsulfonyl)oxy)pyrrolidine-1- carboxylate (149 mg, 0.176 mmol) in DMF (1.8 ml) was added sodium azide (0.014 g, 0.21 1 mmol) and the reaction mixture was stirred at 65 °C for 18h. The reaction was quenched with water and extracted with TBME and the organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as a pale yellow solid in 72% yield; UPLC-MS: Rt = 3.55 mins; MS m/z [M+H]⁺ 794.6; Method E.

Synthesis Example 29.

2C0₃, Kl, acetone, RT, 97%

benzyl 3-( 1 -((tert-butoxycarbonyl)amino)-2-methoxy-2-oxoethylidene)azetidine-1 -

carboxylate

To a solution of benzyl 3-oxoazetidine-1 -carboxylate (5.0 g, 24.4 mmol) and methyl 2- ((tert-butoxycarbonyl)amino)-2-(dimethoxyphosphoryl)acetate (7.24 g, 24.4 mmol) in DCM (122 ml) was added dropwise 2,3,4,6,7,8,9, 10-octahydropyrimido[1 ,2-a]azepine (4.4 ml, 29.2 mmol) and the reaction mixture was stirred at RT for 1 h. The reaction was concentrated to dryness. Water was added and the reaction was made acidic with the addition of 2N HCI aqueous solution. The reaction was then extracted with EtOAc. The organic layer was combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 88% yield; UPLC-MS: Rt = 2.24 mins; MS m/z [M+Na]⁺ 399.1 ; Method E; benzyl 3-(1 -((tert-butoxycarbonyl)amino)-2-methoxy-2-oxoethyl)azetidine-1 -carboxylate

To a solution of benzyl 3-(1 -((tert-butoxycarbonyl)amino)-2-methoxy-2- oxoethylidene)azetidine-1 -carboxylate (8.0 g, 21 .3 mmol) in EtOAc (47 ml) under argon was added 10% palladium on carbon (3.39 g, 3.19 mmol). The atmosphere was replaced by hydrogen and the resulting reaction mixture was stirred at RT for 2h. The reaction was filtered through celite and then concentrated to dryness. The residue was dissolved in

DCM (48 ml) followed by the addition at 0°C of triethylamine (5.9 ml, 42.5 mmol) and benzyl chloroformate (4.8 ml, 31.9 mmol). The reaction was stirred at RT for 72h. The reaction was acidified with a 2N HCI aqueous solution and the reaction was extracted with DCM. The organic layer was combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as an pale yellow solid in 35% yield; UPLC-MS: Rt = 1.03 mins; MS m/z [M+H]⁺ 379.4; Method A.

2-(1-((benzyloxy)carbonyl)azetidin-3-yl)-2-((tert-butoxycarbonyl)amino)acetic acid

To a solution of benzyl 3-(1-((tert-butoxycarbonyl)amino)-2-methoxy-2-oxoethyl)azetidine- 1-carboxylate (2.71 g, 7.16 mmol) in methanol (36 ml) at 0°C was added dropwise a solution of potassium carbonate (1.98 g, 14.3 mmol) in water (36 ml) and the reaction mixture was stirred at RT for 18h. The methanol was removed under reduced pressure and the solution was brought to a pH of ~6 by addition of a 10% HCI aqueous solution. The reaction was then concentrated to dryness. The resulting solid was stirred in 200 ml_ of DCM for 2h at RT and then the solid was removed by filtration. The filtrate was dried over Na₂S0₄, filtered and concentrated to dryness afforded the title compound as a white solid in 89% yield which was used in the next step without further purification; UPLC-MS: Rt = 0.92 mins; MS m/z [M-H]^" 363.2; Method A. benzyl 3-(1-((tert-butoxycarbonyl)amino)-2-(2-(2,5-difluorophenyl)-2-oxoethoxy)-2- oxoethyl) azetidine-1-carboxylate

The product was synthesized in an analogous way as in Example 13, using 2-(1 - ((benzyloxy)carbonyl)azetidin-3-yl)-2-((tert-butoxycarbonyl)amino)acetic acid instead; 97% yield; UPLC-MS: Rt = 2.53 mins; MS m/z [M+Na]⁺ 541 .3; Method E. benzyl 3-(((tert-butoxycarbonyl)amino)(4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)methyl)azetidine-1 -carboxylate

81 % yield; UPLC-MS: Rt = 1 .82 mins; MS m/z [M+H]⁺ 499.3; Method E.

benzyl 3-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl) azetidine-1 -carboxylate

42% yield; UPLC-MS: Rt = 2.71 mins; MS m/z [M+H]⁺ 589.4; Method E.

(R)-benzyl 3-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl)azetidine-1 -carboxylate

The racemic mixture of benzyl 3-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl)azetidine-1 -carboxylate (4.12 g) was submitted to the Separation Laboratory in Basel (contact: Dr. Eric Francotte, Tel. +41 6169 62971 ). The desired enantiomerically enriched (R)-benzyl 3-(amino(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)methyl)azetidine-1 -carboxylate (1 .51 g, 99.3%ee) and undesired (S)- benzyl 3-(amino(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)methyl)azetidine-1 - carboxylate (1 .53 g, 98.0 %ee) were obtained with 74% recovery. The absolute configuration was established by x-ray crystallography of the titled compound.

(R)-benzyl 3-(amino(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)methyl)azetidine-1 - carboxylate

quantitative yield; UPLC-MS: Rt = 1.93 mins; MS m/z [M+H]⁺ 489.3; Method E.

(3R,4R)-tert-butyl 3-((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-

((benzyloxy)carbonyl)azetidin-3-yl)methyl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate

34% yield; UPLC-MS: Rt = 2.27 mins; MS m/z [M+H]⁺ 690.5; Method E.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(1-((benzyloxy)carbonyl)azetidin-3-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-

1-carboxylate

87% yield; UPLC-MS: Rt = 3.05 mins; MS m/z [M+H]⁺ 804.5; Method E. (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-azetidin-3-yl(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(1 -((benzyloxy)carbonyl)azetidin-3- yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate (820 mg, 1 .02 mmol) in methanol (10.2 ml) under argon was added 10% palladium on carbon (54 mg, 0.051 mmol) followed by ammonium formate (772 mg, 12.2 mmol) and the reaction mixture was stirred at RT for 2h. The reaction was filtered over celite and concentrated to dryness. A saturated aqueous solution of NaHC0₃ was added and the reaction was extracted with DCM. The organic layer was combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% MeOH in DCM afforded the title compound as an pale yellow solid in 83% yield; UPLC-MS: Rt = 1 .92 mins; MS m/z [M+H]⁺ 670.4; Method E.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-azetidin-3-yl(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)methyl)-2-hydroxypropanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-azetidin-3-yl(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1- carboxylate (571 mg, 0.853 mmol) in methanol (2.8 ml) at 0°C was added slowly NaBH₄ (213 mg, 5.63 mmol) and the reaction mixture was stirred at 0°C for 7h. The reaction was quenched with the dropwise addition at 0°C of a saturated NH4CI aqueous solution and the reaction was extracted with DCM. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by reverse phase chromatography (5% TFA in MeCN/5% TFA in water) afforded the title compound as an pale yellow solid after lyophilisation as the TFA salt in 35% yield; UPLC-MS: Rt = 1.79 mins; MS m/z [M+H]⁺ 628.0; Method E.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-acetylazetidin-3-yl)(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)methyl)-2-hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

The product was obtained as a by-product of the synthesis of (3R,4R)-tert-butyl 3-(((S)-N- ((R)-azetidin-3-yl(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)methyl)-2- hydroxypropanamido) methyl)-4-fluoropyrrolidine-1-carboxylate; 19% yield; UPLC-MS: Rt = 2.27 mins; MS m/z [M+H]⁺ 670.3; Method E.

Synthesis Example 30.

izyloxy)carbonyl)piperidin-4-yl)-2-((tert-butoxycarbonyl)amino)acetic acid

Prepared as described in WO02076450 A1 , 2002; P. 66. benzyl 4-( 1 -((tert-butoxycarbonyl)amino)-2-(2-(2,5-difluorophenyl)-2-oxoethoxy)-2- oxoethyl)piperidine-1 -carboxylate

The product was synthesized in an analogous way as example 13 using 2-(1 - ((benzyloxy)carbonyl)piperidin-4-yl)-2-((tert-butoxycarbonyl)amino)acetic acid instead; 96% yield; UPLC-MS: Rt = 1 .27 mins; MS m/z [M-H]^" 545.0; Method A. benzyl 4-(((tert-butoxycarbonyl)amino)(4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)methyl)piperidine-1 -carboxylate

103% yield; UPLC-MS: Rt = 1 .22 mins; MS m/z [M-H]^" 525.1 ; Method A. benzyl 4-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl)piperidine-1 -carboxylate

45% yield; UPLC-MS: Rt = 1 .45 mins; MS m/z [M+H]⁺ 617.5; Method A.

(R)-benzyl 4-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl)piperidine-1 -carboxylate

The racemic mixture of benzyl 4-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((tert- butoxycarbonyl)amino)methyl)piperidine-1 -carboxylate (6.00 g) was submitted to the Separation Laboratory in Basel (contact: Dr. Eric Francotte, Tel. +41 6169 62971 ). The desired enantiomerically enriched (R)-benzyl 4-(( 1 -benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)((tert-butoxycarbonyl)amino)methyl)piperidine-1 -carboxylate ( 1 .91 g, >99.5%ee) and undesired (S)-benzyl 4-((1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)((tert-butoxycarbonyl)amino)methyl) piperidine-1 -carboxylate (2.20 g, >99.5 %ee) were obtained with 69% recovery. The absolute configuration was established by x-ray crystallography of the titled compound.

(R)-benzyl 4-(amino( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)methyl)piperidine-1 - carboxylate

quantitative yield; UPLC-MS: Rt = 2.01 mins; MS m/z [M+H]⁺ 517.3; Method E. benzyl 4-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((((3R,4R)-1 -(tert- butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)amino)methyl)piperidine-1 -carboxylate

79% yield; UPLC-MS: Rt = 2.32 mins; MS m/z [M+H]⁺ 718.4; Method E. benzyl 4-((R)-((S)-2-acetoxy-N-(((3R,4R)-1 -(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)methyl)piperidine-1 -carboxylate

76% yield; UPLC-MS: Rt = 3.15 mins; MS m/z [M+H]⁺ 832.5; Method E. (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(piperidin-4-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

56% yield; UPLC-MS: Rt = 1 .94 mins; MS m/z [M+H]⁺ 698.4; Method E.

Synthesis Example 31.

(3S,4S)-tert-butyl 3-(((tert-butyldimethylsilyl)oxy)methyl)-4-(hydroxymethyl)pyrrolidine-1 - carboxylate

Prepared as described in WO06066896, 2006; P. 385-388

(3R,4S)-tert-butyl 3-((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)amino)methyl)-4-(((tert-butyldimethylsilyl)oxy)methyl)pyrroli^

carboxylate

To a solution of (3S,4S)-tert-butyl 3-(((tert-butyldimethylsilyl)oxy)methyl)-4-

(hydroxymethyl) pyrrolidine-1-carboxylate (5.23 g, 15.1 mmol) in dry DCM (38 ml) was added Dess-Martin Periodinane (12.84 g, 30.3 mmol) and the resulting mixture was stirred at RT for 1.5h. This solution was then added to a suspension of (R)-(1-benzyl-4- (2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methanamine (5.80 g, 15.1 mmol) and sodium triacetoxyborohydride (16.0 g, 76.0 mmol) in DCM (38 ml). The resulting mixture was stirred at RT for 18 h. The mixture was then quenched with the addition of water and the reaction mixture was extracted with a 1 M aqueous solution of Na₂S₂0₃ and DCM. The organic layer was washed with a saturated aqueous solution of NaHC0₃ then brine. The organic extractes were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as a yellow oil in 51 % yield; UPLC-MS: Rt = 1.61 mins; MS m/z [M+H]⁺ 71 1.4; Method A.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((tert- butyldimethylsilyl)oxy)methyl) pyrrolidine-1 -carboxylate

To a solution of (3R,4S)-tert-butyl 3-((((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)amino)methyl)-4-(((tert-butyldimethylsilyl)oxy)m pyrrolidine-1 -carboxylate (3.9 g, 5.49 mmol) in DCM (55 ml) at 0°C was added diisopropylethylamine (1 .34 ml, 7.68 mmol) followed by (S)-1 -chloro-1 -oxopropan-2-yl acetate (0.83 ml, 6.58 mmol) and the reaction mixture was allowed to warm to RT and was stirred for 16h. The reaction was extracted with DCM and was washed with saturated aqueous NaHC0₃ solution. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 78% yield; UPLC-MS: Rt = 3.54 mins; MS m/z [M+H]⁺ 825.6; Method E.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(hydroxymethyl)pyrrolidine-1 - carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)- 4-(((tert-butyldimethylsilyl)oxy) methyl) pyrrolidine-1 -carboxylate (3.55 g, 4.30 mmol) in THF (21 .5 ml) was added TBAF (1 .69 g, 6.45 mmol) and the reaction mixture was stirred at RT for 2h. The reaction was diluted with a saturated aqueous NH₄CI solution and the reaction was extracted with DCM and the organic layer was washed with brine. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as an pale yellow oil in 79% yield; UPLC-MS: Rt = 2.42 mins; MS m/z [M+H]⁺ 71 1 .4; Method E.

(3S,4R)-4-(((S)-2-acetoxy-N-((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-1 -(tert-

butoxycarbonyl)pyrrolidine-3-carboxylic acid

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)- 4-(hydroxymethyl)pyrrolidine-1 -carboxylate (300 mg, 0.422 mmol) in acetone (2.1 ml) at 0°C was added dropwise 2 M chromium trioxide in aqueous H₂S0₄ (1 .3 ml, 3.93 mmol) and the reaction mixture was stirred at RT for 1 h. The excess Jones reagent was quenched with the dropwise addition at 0°C of isopropanol (4 ml). The reaction was concentrated to dryness. Water was added and the reaction was extracted with EtOAc. The organic layer was combined, dried over Na2S0₄, filtered and concentrated to dryness afforded the title compound as a solid in 81 % yield which was used in the next step without further purification; UPLC-MS: Rt = 0.65 mins; MS m/z [M+H]⁺ 312.1 ; Method A.

Synthesis Example 32.

tert-butyl 3-(2-(((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut- 3-en-1 -yl)carbamoyl)phenyl)pyrrolidine-1 -carboxylate

To a solution of 2-(1 -(tert-butoxycarbonyl)pyrrolidin-3-yl)benzoic acid (152 mg, 0.523 mmol) in DMF (1 .5 ml) was added HATU (248 mg, 0.653 mmol), diisopropylethylamine (0.22 ml, 1 .31 mmol) and (R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1 -amine (160 mg, 0.435 mmol) and the resulting reaction mixture was stirred at RT for 1 h. The reaction was concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as a diastereomeric mixture as a colorless solid in 77% yield; UPLC-MS: Rt = 2.93 and 2.96 mins; MS m/z [M+H]⁺ 641 .3; Method E. tert-butyl 3-(2-(((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)carbamoyl)phenyl)pyrrolidine-1 -carboxylate

The product was synthesized in an analogous way as Example 14; 67% yield (mixture of diastereoisomers); UPLC-MS: Rt = 2.54 and 2.58 mins; MS m/z [M+H]⁺ 659.3; Method E.

(4R)-4-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-(2-(1 -(tert- butoxycarbonyl)pyrrolidin-3-yl)benzamido)-3,3-dimethylbutanoic acid

28% yield (mixture of diastereoisomers); UPLC-MS: Rt = 2.64 and 2.65 mins; MS m/z [M+H]⁺ 673.1 ; Method E.

Synthesis Example 33. tert-butyl 3-(2-(methoxycarbonyl)phenoxy)azetidine-1 -carboxylate

To a solution of methyl 2-fluorobenzoate (0.83 ml, 6.49 mmol) in DMF (16.2 ml) was added t-butyl 3-hydroxyazetidine-1-carboxylate (1.124 g, 6.49 mmol) and cesium carbonate (10.6 g, 32.4 mmol) and the reaction mixture was stirred at 75°C for 18h. The reaction was extracted with water and DCM. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as a colorless oil in 17% yield; UPLC-MS: Rt = 2.10 mins; MS m/z [M+Na]⁺ ; Method E.

2-((1-(tert-butoxycarbonyl)azetidin-3-yl)oxy)benzoic acid

To a solution of tert-butyl 3-(2-(methoxycarbonyl)phenoxy)azetidine-1-carboxylate (325 mg, 1.06 mmol) in methanol (4.4 ml) and water (0.9 ml) was added lithium hydroxide (127 mg, 5.29 mmol) and the reaction mixture was stirred at RT for 18h. The reaction was extracted with an aqueous 1 M HCI solution and DCM. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the title compound as a colorless solid in 94% yield; UPLC-MS: Rt = 1.71 mins; MS m/z [M+Na]⁺ 330.0; Method E.

(R)-tert-butyl 3-(2-((1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut- 3-en-1-yl)carbamoyl)phenoxy)azetidine-1-carboxylate

The product was synthesized in an analogous way as example 9; 103% yield; UPLC-MS: Rt = 2.95 mins; MS m/z [M+H]⁺ 643.2; Method E.

(R)-tert-butyl 3-(2-((1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)carbamoyl)phenoxy)azetidine-1 -carboxylate

30% yield; UPLC-MS: Rt = 2.53 mins; MS m/z [M+H]⁺ 661 .2; Method E.

(R)-4-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-(2-((1 -(tert- butoxycarbonyl)azetidin-3-yl)oxy)benzamido)-3,3-dimethylbutanoic acid

32% yield; UPLC-MS: Rt = 2.67 mins; MS m/z [M+H]⁺ 675.1 ; Method E.

Synthesis Example 34.

(R)-tert-butyl 3-((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)amino)methyl)pyrrolidine-1-carboxylate

The product was synthesized in an analogous way as example 4; 79% yield; UPLC-MS: Rt = 1.15 mins; MS m/z [M+H]⁺ 566.9; Method A.

(R)-tert-butyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)pyrrolidine-1-carboxylate

91 % yield; UPLC-MS: Rt = 1.25 mins; MS m/z [M+H]⁺ 667.9; Method A.

Additional Payload Examples:

(3R,4S)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-4-hydroxypyrrolidine-

1-carboxylate

To a solution of (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)- 4-hydroxypyrrolidine-1-carboxylate (150 mg, 0.215 mmol) in methanol (1.1 ml) was added potassium carbonate (36 mg, 0.258 mmol) and the reaction mixture was stirred at RT for 18h. The reaction was extracted with a saturated aqueous solution of NH₄CI and DCM. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the title compound as a clear oil in quantitative yield; UPLC-MS: Rt = 1 .20 mins; MS m/z [M+H]⁺ 655.5; Method A.

(S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-2-hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using general protocol 2 for deprotection; 61 % yield; UPLC- MS: Rt = 0.78 mins; MS m/z [M+H]⁺ 555.5; Method A.

(3S,4R)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-4-hydroxypyrrolidine-

1 -carboxylate

The product was generated using an analogous protocol as (3R,4S)-tert-butyl 3-(((S)-N- ((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 2-hydroxypropanamido) methyl)-4-hydroxypyrrolidine-1 -carboxylate; quantitative yield; UPLC-MS: Rt = 1 .19 mins; MS m/z [M+H]⁺ 655.0; Method A. (S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-2-hydroxy-N-(((3R^R)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using general protocol 2 for deprotection; 54% yield; UPLC- MS: Rt = 0.79 mins; MS m/z [M+H]⁺ 555.5; Method A.

(3R,4S)-tert-butyl 3-(((S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylpropyl)-2-hydroxypropanamido)methyl)-4-hydroxypyrrolidine-1 -carboxylate

The product was generated using an analogous protocol as (3R,4S)-tert-butyl 3-(((S)-N- ((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)- 2-hydroxypropanamido) methyl)-4-hydroxypyrrolidine-1 -carboxylate; quantitative yield; UPLC-MS: Rt = 2.88 mins; MS m/z [M+H]⁺ 627.3; Method E.

(S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylpropyl)-2- hydroxy-N-(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)propanamide

The product was deprotected using general protocol 2 for deprotection; 67% yield as a TFA salt; UPLC-MS: Rt = 1 .94 mins; MS m/z [M+H]⁺ 527.2; Method E. ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 7.88-7.87 (1 H, m), 7.81 -7.76 (1 H, m), 7.41 -7.28 (7H, m), 7.12-7.06 (1 H, m), 5.80 (1 H, s), 5.59-5.58 (1 H, m), 5.34-5.30 (1 H, m), 5.05-4.95 (2H, m), 4.72-4.65 (1 H, m), 3.93-3.87 (1 H, m), 3.75—3.66 (2H, m), 3.08-3.03 (1 H, m), 2.73-2.64 (1 H, m), 2.18-2.14 (1 H, m), 1 .75-1 .58 (1 H, m), 1 .30-1 .28 (3H, m), 0.80 (9H, s).

((pentylcarbamoyl)oxy)pyrrolidine-1 -carboxylate

To a solution of (S)-1-(((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)(((3S,4S)-4-hydroxypyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate (4.6 mg, 0.0066 mmol) and diisopropylethylamine (0.001 1 ml, 0.066 mmol) in dry DMF (0.07 ml) was added bis(4-nitrophenyl) carbonate (4.5 mg, 0.015 mmol) and the reaction mixture was stirred at RT for 1 h. Pentylamine (0.003 ml, 0.027 mmol) was added and the reaction was stirred for 1 .5 h at RT. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the title compound as a pale yellow solid in 99% yield; UPLC-MS: Rt = 1 .16 mins; MS m/z [M+H]⁺ 810.5; Method E.

(3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl pentylcarbamate

The product was deprotected using general protocol 1 for deprotection; 43% yield; UPLC- MS: Rt = 1 .96 mins; MS m/z [M+H]⁺ 668.5; Method E; ¹ H-NMR (CDCI₃, 400 MHz, mixture of rotomers): δ 9.71 (2H, br), 7.72-7.67 (1 H, m), 7.40-7.13 (7H, m), 7.04-6.92 (1 H, m), 6.85-6.78 (1 H, m), 5.67-5.64 (1 H, m), 5.32-4.86 (3H, m), 4.65-4.38 (1 H, m), 3.89-1 .72 (15H, m), 1 .51 -1 .07 (13H, m), 0.84-0.58 (3H, m).

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(pentylcarbamoyl)pyrrolidine-

1 -carboxylate

To a solution of pentylamine (0.023 ml, 0.1 19 mmol) in DCM (0.18 ml) was added HATU

(88mg, 0.232 mmol) followed by a solution of (3S,4R)-4-(((S)-2-acetoxy-N-((R)-(1 -benzyl-

4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)propanamido)methyl)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (80 mg, 0.1 10 mmol) in DMF (0.18 ml) and the reaction mixture was stirred at RT for 1 h. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na₂SC>4, filtered and concentrated to dryness to afforded the title compound as a pale yellow solid in quantitative yield; UPLC-MS: Rt = 1 .35 mins; MS m/z [M+H]⁺ 794.2; Method E.

(3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-N-pentylpyrrolidine-3-carboxamide

The product was deprotected using general protocol 1 for deprotection; 43% yield as a TFA salt; UPLC-MS: Rt = 0.94 mins; MS m/z [M+H]⁺ 652.5; Method A; ¹ H-NMR (CDCI₃, 400 MHz, mixture of rotomers): δ 10.31 (1 H, br), 8.78 (1 H, br), 7.94-7.66 (1 H, m), 7.51 - 7.42 (1 H, m), 7.32-7.24 (2H, m), 7.17-7.15 (2H, m), 7.01 -6.91 (2H, m), 6.84-6.80 (1 H, m), 5.61 -5.58 (1 H, m), 5.20-5.16 (1 H, m), 5.05-5.01 (1 H, m), 4.67-4.49 (1 H, m), 3.86-3.83 (1 H, m), 3.63-2.54 (14H, m), 2.05-1 .96 (1 H, m), 1 .44-1 .36 (2H, m), 1 .32-1 .13 (10H, m), 0.83- 0.80 (3H, m).

(R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)pyrrolidine-1-carboxylate

The product was synthesized in an analogous way as example 1 using (R)-tert-butyl 3- ((((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)amino)methyl) pyrrolidine-1-carboxylate; 59% yield; UPLC-MS: Rt = 1.36 mins; MS m/z [M+H]⁺ 680.9; Method A.

(R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidine-1-carboxylate

88% yield; UPLC-MS: Rt = 2.55 mins; MS m/z [M+H]⁺ 639.4; Method E.

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-2-hydroxy-N-((S)-pyrrolidin-3-ylmethyl)propanamide

The product was deprotected using general protocol 2 for deprotection; 50 equiv.; 70% yield; UPLC-MS: Rt = 0.81 mins; MS m/z [M+H]⁺ 538.8; Method A; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 7.87-7.82 (1H, m), 7.51-7.52 (1H, m), 7.40-7.23 (4H, m), 7.09-7.03 (2H, m), 6.92-6.86 (1H, m), 5.75-5.72 (1H, m), 5.36-5.32 (1H, m), 5.10-5.06 (1H, m), 4.57-4.52 (1H, m), 3.99-3.96 (1H, m), 3.80-3.76 (1H, m), 3.52-3.21 (4H, m), 2.97-2.91 (1H, m), 2.78-2.69 (2H, m), 2.55-2.50 (1H, m), 2.08-2.04 (1H, m), 1.67-1.60 (2H, m), 1.50- 1.47 (1H, m), 1.42-1.28 (6H, m), 0.90-0.80 (1H, m).

1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-((S)-1-hydroxypropan-2-yl)-1-((S)-pyrrolidin-3-ylmethyl)urea

The product was deprotected using general protocol 2 for deprotection; 50 equiv. TFA; 60% yield; UPLC-MS: Rt = 0.82 mins; MS m/z [M+H]⁺ 584.3; Method A; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 7.84-7.82 (1H, m), 7.47-7.46 (1H, m), 7.35-7.24 (5H, m), 7.05-6.99 (1H, m), 6.85-6.84 (1H, m), 5.92 (1H, s, br), 5.50-5.43 (2H, m), 5.10-5.03 (1H, m), 3.99-2.07 (17H, m), 1.64-0.75 (9H, m). (S)-N-((R)-(1 -acetylazetidin-3-yl)(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)m N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The product was deprotected using general protocol 2 for deprotection; 50 equiv.; 45% yield; UPLC-MS: Rt = 1.45 mins; MS m/z [M+H]⁺ 570.1 ; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 9.07 (1 H, s, br), 8.72 (1 H, s, br), 7.83-7.66 (2H, m), 7.44- 7.28 (5H, m), 7.15-7.10 (1 H, m), 7.03-6.98 (1 H, m), 6.23-5.65 (1 H, m), 5.52-5.10 (3H, m), 4.54-4.44 (1 H, m), 4.34-2.69 (1 1 H, m), 2.44-2.1 1 (2H, m), 1 .70-1 .55 (3H, m), 1 .30-0.98 (3H, m).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1 -

(methoxycarbonyl)azetidin-3-yl)methyl)-2-hydroxypropanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-N-((R)-azetidin-3-yl(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)methyl)-2-hydroxypropanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate (20 mg, 0.027 mmol) in DCM (0.27 ml) was added methyl chloroformate (0.0042 ml, 0.054 mmol), triethylamine (0.015 ml, 0.108 mmol) and DMAP (0.3 mg, 0.003 mmol) and the reaction mixture was stirred at RT for 1 .5h. The reaction was extracted with a saturated aqueous solution of NaHC0₃ and DCM. The organic layers were combined, dried over Na2SC>4, filtered and concentrated to dryness to afforded the title compound as a colorless solid in 124% yield; UPLC-MS: Rt = 2.51 mins; MS m/z [M+H]⁺ 686.2; Method E.

methyl 3-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)methyl)azetidine-1 -carboxylate

The product was deprotected using general protocol 2 for deprotection; 50 equiv.; 48% yield; UPLC-MS: Rt = 1 .63 mins; MS m/z [M+H]⁺ 586.1 ; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 9.05 (1 H, s, br), 8.65 (1 H, s, br), 7.83-7.66 (2H, m), 7.43- 7.29 (5H, m), 7.15-7.10 (1 H, m), 7.00-6.98 (1 H, m), 6.24-5.71 (1 H, m), 5.51 -5.09 (3H, m), 4.52-4.43 (1 H, m), 4.22-2.76 (14H, m), 2.47-2.10 (2H, m), 1.29-0.97 (3H, m).

4-nitrophenyl 4-((R)-((S)-2-acetoxy-N-(((3R,4R)-1 -(tert-butoxycarbonyl)-4-fluoropyrrolidin-

3-yl)methyl)propanamido)(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)methyl)piperidine-1 -carboxylate

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(piperidin-4-yl)methyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (80 mg, 0.1 15 mmol) in dry DMF (0.57 ml) was added diisopropylethylamine (0.20 ml, 1.15 mmol) and bis(4-nitrophenyl) carbonate (70 mg, 0.229 mmol) and the reaction mixture was stirred at RT for 1 h. The reaction was extracted with water and EtOAc. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness. . Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded as a pale yellow solid in 138% yield; UPLC-MS: Rt = 1.44 mins; MS m/z [M+H]⁺ 863.0; Method A.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-

(pentylcarbamoyl)piperidin-4-yl)methyl)-2-hydroxypropanamido)methyl)-4- fluoropyrrolidine-1-carboxylate

A solution of 4-nitrophenyl 4-((R)-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)methyl)piperidine-1-carboxylate (120 mg, 0.139 mmol) and pentylamine (0.024 ml, 0.209 mmol) in MeOH (0.7 ml) was heated to 100 °C for 1 h in a microwave. Additional pentylamine was added (0.048 ml, 0.417 mmol) added and the reaction mixture was heated again to 100 °C for 1 h in a microwave. The cycle was repeated 3 additional times until the reaction was completed. Potassium carbonate (23 mg, 0.167 mmol) was then added and the reaction was stirred at RT for 18h. The reaction was then extracted with a saturated aqueous solution of NH₄CI and DCM then washed with a saturated aqueous solution of NaHC0₃. The organic layers were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the title compound as a yellow solid in 60% yield; UPLC-MS: Rt = 2.75 mins; MS m/z [M+H]⁺ 769.6; Method E. 4-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)methyl)-N-pentylpiperidine- carboxamide

The product was deprotected using general protocol 2 for deprotection; 50 equiv.; 50% yield as a TFA salt; UPLC-MS: Rt = 1 .86 mins; MS m/z [M+H]⁺ 669.4; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 9.1 1 -9.02 (1 H, m), 8.84 (1 H, br), 7.86-7.68 (2H, m), 7.41 -7.38 (2H, m), 7.35-7.31 (1 H, m), 7.28-7.26 (1 H, m), 7.15-7.07 (2H, m), 6.35-6.32 (1 H, br), 5.62-5.02 (4H, m), 4.54-4.36 (1 H, m), 4.01 -3.18 (9H, m), 2.95-2.94 (2H, m), 2.69-2.02 (4H, m), 1 .45-1 .17 (1 1 H, m), 0.94-0.84 (5H, m), 0.49-0.40 (1 H, m).

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -acetylpiperidin-4-yl)(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -

carboxylate

The product was generated by an analogous reaction to the formation of (S)-N-((R)-(1 - acetylazetidin-3-yl)(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)methyl)-N-(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide; quantitative yield; UPLC-MS: Rt = 2.53 mins; MS m/z [M+H]⁺ 740.5; Method A. (S)-N-((R)-(1 -acetylpiperidin-4-yl)(1 -^

yl)methyl)-N-(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The product was deprotected using general protocol 1 for deprotection; 37% yield; UPLC- MS: Rt = 1 .46 mins; MS m/z [M+H]⁺ 598.4; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 8.97-8.80 (1 H, m), 8.63-8.53 (1 H, m), 7.79-7.62 (2H, m), 7.34-6.99 (7H, m), 5.59-5.44 (1 H, m), 5.33-4.95 (3H, m), 4.45-1 .98 (14H, m), 1 .88-1 .79 (3H, m), 1 .47-0.26 (7H, m).

ethyl 4-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((S)-N-(((3R,4R)-1 -(tert- butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)methyl)piperidine-

1 -carboxylate

The product was generated by an analogous reaction to the formation of (3R,4R)-tert- butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1 -

(methoxycarbonyl)azetidin-3-yl)methyl)-2-hydroxypropanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate using ethyl chloroformate instead; quantitative yield;

UPLC-MS: Rt = 2.94 mins; MS m/z [M+H]⁺ 770.5; Method E. ethyl 4-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)((S)-N-(((3S,4 )-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)methyl)piperidine-1 -carboxylate

The product was deprotected using general protocol 1 for deprotection; 24% yield; UPLC- MS: Rt = 1 .78 mins; MS m/z [M+H]⁺ 628.5; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotomers): δ 9.04-8.96 (1 H, m), 8.68-8.59 (1 H, m), 7.87-7.70 (2H, m), 7.42-7.07 (7H, m), 5.64-5.52 (1 H, m), 5.41 -5.02 (3H, m), 4.53-4.35 (1 H, m), 4.03-3.18 (1 1 H, m), 2.86-2.57 (3H, m), 2.27-2.06 (1 H, m), 1 .51 -1 .37 (1 H, m), 1 .31 -0.40 (9H, m).

4-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2-one

To a solution of 4-(hydroxymethyl)pyrrolidin-2-one (1 .00 g, 8.69 mmol) in DCM (80 ml) was added imidazole (887 mg, 13.0 mmol) and tert-butylchlorodimethylsilane (1 .57 g, 10.4 mmol) and the reaction mixture was stirred at RT for 3h. The reaction was concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded as a clear oil in 101 % yield; UPLC-MS: Rt = 1 .95 mins; MS m/z [M+H]⁺ 230.0; Method E. izyloxy)methyl)-4-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2-one

To a solution of 4-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2-one (2.00 g, 8.72 mmol) in THF (29 ml) was added sodium hydride (384 mg, 9.59 mmol) and the reaction was stirred at RT for 30 mins. ((Chloromethoxy)methyl)benzene (2.05 g, 13.1 mmol) was then added and the reaction mixture was stirred at RT for 18h. The reaction was quenched with a saturated aqueous solution of NH₄CI and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded as a clear oil in 1 1 % yield; UPLC-MS: Rt = 2.79 mins; MS m/z [M+Na]⁺ 372.2; Method E.

izyloxy)methyl)-4-(hydroxymethyl)pyrrolidin-2-one

To a solution of 1-((benzyloxy)methyl)-4-(((tert-butyldimethylsilyl)oxy)methyl)pyrrolidin-2- one (340 mg, 973 mmol) in THF (3.2 ml) was added TBAF (254 mg, 0.973 mmol) and the reaction mixture was stirred at RT for 1.5h. The reaction was quenched with a saturated aqueous solution of NH₄CI and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded as a clear oil in 90% yield; ¹H-NMR (CDCI₃, 400 MHz): δ 7.55-7.7.27 (5H, m), 4.89-4.85 (2H, s), 4.55-4.52 (2H, s), 3.65-3.53 (3H, m), 3.35-3.31 (1 H, m), 2.56-2.48 (2H, m), 2.27-2.20 (1 H, m). 4-((((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)amino)methyl)-1 -((benzyloxy)methyl)pyrrolidin-2-one

The product was synthesized in an analogous way as example 1 using 1 - ((benzyloxy)methyl)-4-(hydroxymethyl)pyrrolidin-2-one to give the product as a mixture of diastereoisomers; 36% yield; UPLC-MS: Rt = 1 .88 mins; MS m/z [M+H]⁺ 601 .4; Method E.

(2S)-1 -(((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)((1 -((benzyloxy)methyl)-5-oxopyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl

acetate

The product was synthesized in an analogous way as example 1 ; 35% yield; UPLC-MS: Rt = 2.59 mins; MS m/z [M+H]⁺ 715.5; Method E.

(2S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-N-((1 -((benzyloxy)methyl)-5-oxopyrrolidin-3-yl)methyl)-2-hydroxypropanam

To a solution of (2S)-1 -(((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)((1 -((benzyloxy)methyl)-5-oxopyrrolidin-3-yl)methyl)amino)-1 - oxopropan-2-yl acetate (72 mg, 0.101 mmol) in methanol (1 .0 ml) was added potassium carbonate (21 mg, 20.9 mmol) and the reaction mixture was stirred at RT for 0.5h. The reaction was quenched with a saturated aqueous solution of NH₄CI and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness to afforded the product as a pale yellow solid in 95% yield; UPLC-MS: Rt = 1 .21 mins; MS m/z [M+H]⁺ 673.3; Method A.

(2S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-2-hydroxy-N-((5-oxopyrrolidin-3-yl)methyl)propanamide

To a solution of (2S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-

2H-pyran-4-yl)methyl)-N-((1 -((benzyloxy)methyl)-5-oxopyrrolidin-3-yl)methyl)-2- hydroxypropanamide (65 mg, 0.096 mmol) in DCM (1 .0 ml) was added dropwise a 1 .25 M

HCI solution in MeOH (1 .22 ml, 1 .53 mmol) and the reaction mixture was stirred at 60 °C for 18h. The reaction was quenched with a saturated aqueous solution of NaHC0₃ and extracted with DCM. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound in 13% yield; UPLC-MS: Rt = 0.97 mins; MS m/z [M+H]⁺ 553.4; Method A; ¹H-NMR (CDCI₃, 400 MHz, mixture of rotomers): δ 7.76-7.70 (1 H, m), 7.45-7.42 (1 H, m), 7.29-7.24 (3H, m), 7.14-7.13 (2H, m), 7.00-6.92 (1 H, m), 6.83-6.79 (1 H, m), 5.66-5.63 (1 H, m), 5.25-5.19 (1 H, m), 5.01- 4.95 (1 H, m), 4.33-4.26 (1 H, m), 3.90-3.87 (1 H, m), 3.71-3.68 (1 H, m), 3.54-3.08 (5H, m), 2.91-2.74 (1 H, m), 2.68-2.59 (1 H, m), 2.35-0.71 (8H, m).

(R)-tert-butyl 3-(2-((1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethyl-4- oxo-4-(pentylamino)butyl)carbamoyl)phenoxy)azetidine-1-carboxylate

To a solution of (R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-(2-((1-(tert- butoxycarbonyl)azetidin-3-yl)oxy)benzamido)-3,3-dimethylbutanoic acid (30 mg, 0.044 mmol) in DMF (0.45 ml) was added HATU (20.3 mg, 0.053 mmol), diisopropylethylamine (0.04 ml, 0.222 mmol) and pentylamine (0.006 ml, 0.049 mmol) and the reaction mixture was stirred at RT for 1 h. Additional HATU (8.5 mg, 0.022 mmol), diisopropylethylamine (0.023 ml, 0.133 mmol) and pentylamine (0.005 ml, 0.044 mmol) and the reaction mixture was stirred at RT for 0.5h. The reaction was extracted with a 2M aqueous solution of Na2CC>3 and EtOAc. The organic layers were combined, dried over Na2SC>4, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% EtOAc in heptane afforded the title compound as a colorless solid in 109% yield; UPLC-MS: Rt = 2.72 mins; MS m/z [M+H]⁺ 744.3; Method E. (R)-2-(azetidin-3-yloxy)-N-(1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-2,^ dimethyl-4-oxo-4-(pentylamino)butyl)benzamide

The product was deprotected using general protocol 2 for deprotection; 20 equiv. TFA; 83% yield (TFA salt); UPLC-MS: Rt = 1.96 mins; MS m/z [M+H]⁺ 644.2; Method E; ¹H-NMR (DMSO, 400 MHz): δ 9.01-9.00 (1H, m), 8.94-8.85 (2H, m), 7.97-7.94 (1H, m), 7.78-7.74 (1H, m), 7.65-7.63 (1H, m), 7.59-7.58 (1H, m), 7.46-7.42 (1H, m), 7.38-7.24 (6H, m), 7.11- 7.06 (2H, m), 6.82-6.80 (1H, m), 5.56-5.39 (3H, m), 5.18-5.12 (1H, m), 4.50-4.46 (1H, m), 4.40-4.36 (1H, m), 4.30-4.24 (1H, m), 4.18-4.12 (1H, m), 3.10-2.93 (2H, m), 2.77-2.74 (1H, m), 2.23-2.20 (1H, m), 1.38-1.31 (2H, m), 1.24-1.19 (4H, m), 1.09 (3H, s), 0.94 (3H, s), 0.83-0.79 (3H, m).

Synthesis Example 35. Benzyl group modifications.

(R)-1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1 -amine, TFA salt

A solution of 1 1 g (R)-tert-butyl (1 -(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1 -yl)carbamate and 9ml_ TFA in 150mL DCM was stirred at room temperature for 16h. The reaction mixture was concentrated under vacuum and 18.8g of a brown/green oil was obtained. This material was taken forward directly in the next step (assuming 100% conversion and purity of 48.5%). UPLC-MS: Rt = 0.73 mins; MS m/z [M+H]⁺ 278.2; Method A.

(3R.4R)-benzyl 3- R)-1-(4-(2.5-difluorophenyl)-1 H-imidazol-2-yl)-2.2-dimethylbut-3- en-1 -yl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate

Step 1 : A stirring solution of (3R,4S)-benzyl 3-fluoro-4-(hydroxymethyl)pyrrolidine-1- carboxylate (8.28g) in dry DCM (100mL) was placed under an argon atmosphere and the temperature of the solution kept constant by the use of a water bath containing ambient temperature water. (In similar reactions, temperature control was found to be important: if the temperature of the reaction mixture is a few degrees hotter, elimination of the fluoro group occurs to some extent. Similarly, if the reaction is run at cooler temperatures (0°C - 10°C) then the oxidation reaction is not fast enough.) Dess-Martin Periodinane (15.85g) was then added in one portion and the reaction mixture stirred at (approximately) 18°C for 20 minutes - and then taken directly to step 2.

Step 2 : The reaction mixture from Step 1 was filtered - through a 0.45um PTFE syringe filter - into a stirring solution of (R)-1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1 -amine, TFA salt (18.84g) and sodium triacetoxyborohydride (19.8g) in dry DCM (150ml_) Note : This solution was previously ultrasonicated to disperse and solubilise any large lumps of triacetoxyborohydride. Stirring was continued under an argon atmosphere - whilst ensuring that the temperature did not exceed 20°C (by using a tap water bath). After 2 hours, LC-MS showed that the reaction was virtually finished with 31 % conversion to desired product. The reaction was quenched with the addition of de- ionised water (150ml_) and the resulting slurry stirred vigorously at RT for 1 hour. The resultant inorganic solids were filtered through No. 1 Filter paper and the filtrate slowly made basic with the portionwise addition of sodium bicarbonate - whilst also vigorously stirring. Once the addition of sodium bicarbonate was complete (no more effervsecence observed), the biphasic solution was transferred to a separating funnel and the layers separated. The aqueous layer was re-extracted with dichloromethane (2x100ml_). The combined organics were washed with sat. bicarbonate (150ml), sat. brine (150ml_), dried over MgSC>4, filtered through No.1 filter paper and concentrated in vacuo to give a dark brown oil. The material was purified directly as follows:

System : Biotage SP4 Normal Phase Purification System

Stationary Phase: 40+M Silica Cartridge

Binary Solvent System: Polar Solvent : 20% v/v methanol/dichloromethane, Non-Polar Solvent : dichloromethane

Gradient : 0% non-polar/polar solvent maintained for 25 minutes and then ramped to 40% polar solvent over a period of 35 minutes - and then maintained at this level for a further 15 minutes - by which time the product had already eluted from the column. Product containing fractions were combined and concentrated in vacuo to give 12.65g of a dark brown glass-like oil, approx 47% pure by LCMS. UPLC-MS: Rt = 1.15 min; MS m/z

[M+H]⁺ 514.3; Method A.

This material was taken forward directly to the next step.

(3R.4R)-benzyl 3-((((Ι¾)-1 -(4-(2.5-difluorophenyl)-1 -(3-(methoxymethoxy)benzyl)-1 H- imidazol-2-yl)-2,2-dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1- carboxylate

To a solution of (3R,4R)-benzyl 3-((((R)-1-(4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylbut-3-en-1-yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate (12.65g) and 3-(methoxymethoxy)benzyl methanesulfonate (8.57g, prepared according to

Bioorg.Med.Chem. 2006, 14, 1771-1784) in anhydrous DMF (100ml) at room temperature and under an atmosphere of argon, was added potassium carbonate (8.02g). A water condenser was fitted and the mixture was stirred at 75°C for 3 hours - by which time LCMS showed that the reaction was complete. The reaction mixture was cooled to room temperature and partitioned between de-ionised water (120ml_) and ethyl acetate (120ml). After extraction, the aqueous phase was re-extracted with ethyl acetate (150ml_). The combined organics were then washed with sat. brine (120ml_), dried over MgSC>4, filtered through No.1 filter paper and concentrated in vacuo to give a dark brown oil. Crude Yield : 19.88g. The material was purified directly as follows:

System : Biotage SP4 Normal Phase Purification System

Stationary Phase: 40+M Silica Cartridge

Binary Solvent System: Polar Solvent : ethyl acetate, Non-Polar Solvent : n-heptane

Stepwise Gradient :

1 . 0% Polar solvent - ^■> Maintained for 5 Minutes

2. 0% Polar Solvent - -> Ramped to 5% Polar Solvent over 15 Minutes

3. 5% Polar solvent - ^■> Maintained for 10 Minutes

4. 5% Polar Solvent - -> Ramped to 7% Polar Solvent over 10 Minutes

5. 7% Polar solvent - ^■> Maintained for 20 Minutes

6. 7% Polar Solvent - -> Ramped to 12% Polar Solvent over 10 Minutes

7. 12% Polar solvent -> Maintained for 3 Minutes

8. 12% Polar Solvent -> Ramped to 20% Polar Solvent over 10 Minutes

9. 20% Polar solvent -> Maintained for 10 Minutes

10. 20% Polar Solvent -> Ramped to 25% Polar Solvent over 10 Minutes

1 1 . 25% Polar solvent -> Maintained for 30 Minutes

The desired product started eluting from the column between 7% Polar Solvent and 12% Polar Solvent. The product containing fractions were combined and concentrated in vacuo to give a brown oil. Yield : 7.7g - 65% Pure by LC-MS. UPLC-MS: Rt = 1 .25 min; MS m/z [M+H]⁺ 664.5; Method A.

This material was taken forward directly to the next step

(3R.4R)-benzyl 3-«((R)-1 -(4-(2.5-dif luorophenylH -(3-fluoro-5- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate

Was prepared analogous to (3R,4R)-benzyl 3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)amino)methyl)-4-fluoropyrrolidine-1 -carboxylate

UPLC-MS: Rt = 5.73 min; Method B.

This material was taken forward directly to the next step

(3R.4R)-benzyl 3-(«S)-2-acetoxy-N-((R)-1 -(4-(2.5-dif luorophenylH -(3-

(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

A solution of (3R,4R)-benzyl 3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-

(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate_(3.85g) in anhydrous DCM (50ml_) was cooled to 0°C with an ice bath. To this solution was addded DIPEA (1.978ml_) and (S)-1-Chloro-1-oxopropan-2-yl acetate (0.956ml_, Fluka) - cloudy fumes observed. The dark-orange coloured reaction mixture was allowed to warm to room temperature and stirring continued for 1 hour - by which time LC-MS showed reaction to be complete with conversion to desired product. The volatiles were removed under high vacuum and the crude material purified directly as follows:

System : Biotage SP4 Normal Phase Purification System

Stationary Phase: 40+M Silica Cartridge

Binary Solvent System: Polar Solvent : ethyl acetate, Non-Polar Solvent : n-heptane Stepwise Gradient :

1. 0% Polar solvent -> Maintained for 10 Minutes

2. 0% Polar Solvent -> Ramped to 46% Polar Solvent over 20 Minutes

3. 46% Polar solvent -> Maintained for 30 Minutes - By this stage, all desired product had already eluted from the column. The run was stopped at this point.

Product containing fractions were combined and concentrated in vacuo to give a brown oil Yield : 2.545g - 95% Pure by LC-MS. UPLC-MS: Rt = 1.49 min; MS m/z [M+H]⁺ 777.5; Method A.

(3R.4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1 -(4-(2.5-difluorophenyl)-1 -(3-fluoro-5-

A solution of (3R,4R)-benzyl 3-((((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-

(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1- yl)amino)methyl)-4-fluoropyrrolidine-1-carboxylate_(1.2g) in anhydrous DCM (20ml_) was cooled to 0°C with an ice bath. To this solution was addded DIPEA (0.822ml_) and

(S)-1-Chloro-1-oxopropan-2-yl acetate (0.397ml_, Fluka) - cloudy fumes observed. The dark-orange coloured reaction mixture was allowed to warm to RT and stirring continued for 1 hour - by which time LC-MS showed reaction to be complete with conversion to desired product. The reaction mixture was then partitioned between de-ionised water ( 100ml_) and dichloromethane (70ml). After extraction, the aqueous phase was re- extracted with dichloromethane (70ml_). The combined organics were then washed with sat. brine ( 100ml_), dried over MgSC>4, filtered through No.1 filter paper and concentrated in vacuo to give a dark orange-coloured oil. Yield: 1 .4g UPLC-MS: Rt = 1 .46 min; MS m/z [M+H]⁺ 796.5; Method A.

The material was of suitable purity to be used directly in the next step without need for further purification.

(3R.4R)-benzyl 3-«(S)-2-acetoxy-N-((R)-1 -(4-(2.5-dif luorophenylH -(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hvdroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

A solution of (3R,4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1 -(4-(2,5-difluorophenyl)-1 -(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1 - yl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate_(2.545g) in anhydrous THF (25ml_) was cooled to 0°C with an ice bath and then treated with the dropwise addition of 1 .OM Borane THF complex (12.45ml_). The reaction mixture was allowed to warm to room temperature and stirring continued for 4 hours. The reaction mixture was then cooled to 0°C and the excess borane was quenched with the sequential addition of a 1 : 1 mixture THF:EtOH (70ml_), pH 7.0 Phosphate buffer solution (90ml_) and 30% hydrogen peroxide solution (4.45ml_). The reaction mixture was left stirring at room temperature for 16 hours and then sat. brine ( 15ml) added to the mixture. The mixture was transferred to a separating funnel and extracted with ethyl acetate (2x200ml). The combined organics were washed with 20% w/v Na2S20s (3x80ml), sat. brine (150ml), dried over MgSC>4, filtered through No. 1 filter paper and concentrated in vacuo to give a light brown wax-like solid. Yield : 2.5917g - 61 % Pure by LC-MS. The material was taken forward to the next step without need for purification. UPLC-MS: Rt = 1 .29 min; MS m/z [M+H]⁺ 795.6; Method A.

(3R.4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1 -(4-(2.5-difluorophenyl)-1 -(3-fluoro-5- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hvdroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

A solution of (3R,4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1 -(4-(2,5-difluorophenyl)-1 -(3- fluoro-5-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-2,2-dimethylbut-3-en-1 - yl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate (1 .4g) in anhydrous THF (25ml_) was placed under an argon atmosphere and cooled to 0°C with an ice bath. It was then treated with the dropwise addition of 1 .0M Borane THF complex (6.27ml_). The reaction mixture was allowed to warm to room temp and stirring continued for 4 hours. The mixture was then cooled to 0°C and the excess borane was quenched with the sequential addition of a 1 : 1 mixture THF:EtOH (70ml_), pH 7.0 Phosphate buffer solution (90ml_) and 30% hydrogen peroxide solution (2.24ml_). The mixture was left stirring at room temp for 16 hours and then sat. brine (15ml) added to the mixture. The mixture was transferred to a separating funnel and extracted with ethyl acetate (2x200ml). The combined organics were washed with 20% w/v Na2S2C>5 (2x80ml), sat. brine (150ml), dried over MgSC>4, filtered through No. 1 filter paper and concentrated in vacuo to give a light brown wax-like solid._Yield : 1 .445g - 74% Pure by LC-MS. The material was taken forward to the next step without need for purification. UPLC-MS: Rt = 1 .28 min; MS m/z [M+H]⁺ 814.7; Method A.

(S)-1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3-(methoxymethoxy)benzyl)-1 H-imidazol-2- yl)-4-hvdroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1- oxopropan-2-yl acetate

To a solution of (3R,4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3- (methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate_(2.59g) in methanol (50ml) - which had been purged with argon - was added ammonium formate (2.508g) and 5 wt% palladium on carbon (423mg). The resulting black suspension was stirred under an argon atmosphere at 50°C for 2 hours - by which time LC-MS showed that the reaction was complete. The reaction mixture was filtered through a No. 1 filter paper - and the palladium catalyst filter cake washed with methanol (2x30ml) and dichloromethane (30ml). The filtrate was then carried forward directly to the next step. (It was assumed 100% conversion at a purity of 60%.) UPLC-MS: Rt = 0.85 min; MS m/z [M+H]⁺ 661.8; Method A.

(S)-1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-(methoxymethoxy)benzyl)-1 H- imidazol-2-yl)-4-hvdroxy-2,2-dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)amino)-1 -oxopropan-2-yl acetate To a solution of (3R,4R)-benzyl 3-(((S)-2-acetoxy-N-((R)-1-(4-(2,5-difluorophenyl)-1-(3- fluoro-5-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (1 445g) in methanol (20ml) - which had been purged with argon - was added ammonium formate (2.489g) and 5 wt% palladium on carbon (280mg). The resulting black suspension was stirred under an argon atmosphere at 50°C for 1.5 hours - by which time LC-MS showed that the reaction was complete. The reaction mixture was filtered through a No. 1 filter paper - and the palladium catalyst filter cake washed with methanol (2x30ml) and dichloromethane (30ml). The filtrate was then carried forward directly to the next step. (It was assumed 100% conversion at a purity of 76%).

(3R.4R)-tert-butyl 3-(«S)-2-acetoxy-N-«R)-1 -(4-(2.5-difluorophenyl)-1 -(3- (methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hvdroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

To the reshly prepared filtrate containing (S)-1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)(((3S,4R)- 4-fluoropyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl acetate was added additional methanol (50ml_) and BOC Aanhydride (0.864g). The reaction mixture was stirred at RT for 30 minutes - by which time LC-MS showed that the pyrrolidine BOC-protection was complete. The volatiles were concentrated in vacuo and the residue partioned between ethyl acetate (150ml_) and de-ionised water (100ml_).The mixture was transferred to a separating funnel and, after extraction, the aqueous phase was re-extracted with ethyl acetate (100ml). Note : During the extraction process, an emulsion formed. This was resolved by adding a small quantity of sat. brine to the separating funnel. The combined organics were then washed with sat. brine (150ml), dried over MgSC>4, filtered through No.

1 filter paper and concentrated in vacuo to give a virtually clear & colourless oil. Crude yield : 2.695g 56% Pure by LC-MS @ 254nm. Purification was performed as follows:

System : Biotage SP4 Normal Phase Purification System Stationary Phase: 25+M Silica Cartridge

1. 0% Polar solvent -> Maintained for 5 Minutes

2. 0% Polar Solvent -> Ramped to 80% Polar Solvent over 30 Minutes

3. 80% Polar solvent -> Maintained for 25 Minutes

4. 80% Polar Solvent -> Ramped to 100% Polar Solvent over 10 Minutes

5. 100% Polar solvent -> Maintained for 15 Minutes

Product containing fractions were combined and concentrated under high vacuum to give a white foam. Yield : 1.25g, 87% Pure by LC-MS @ 254nm. UPLC-MS: Rt = 1.30 min; MS m/z [M+H]⁺ 761.6; Method A.

(3R.4R)-tert-butyl 3-( S)-2-acetoxy-N-((R)-1-(4-(2.5-difluorophenyl)-1-(3-fluoro-5-

(methoxymethoxy)benzyl)-1H-imidazol-2-yl)-4-hvdroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

To the reshly prepared filtrate containing (S)-1-(((R)-1-(4-(2,5-difluorophenyl)-1-(3- fluoro-5-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1 -oxopropan-2-yl acetate was added additional methanol (20ml_) and BOC anhydride (0.767g). The reaction mixture was stirred at room temp for 1 hour - by which time LC-MS showed that the pyrrolidine BOC-protection was complete. The volatiles were concentrated in vacuo and purified directly as follows:

System : Biotage SP4 Normal Phase Purification System

Stationary Phase: 25+M Silica Cartridge

1. 0% Polar solvent -> Maintained for 5 Minutes

2. 0% Polar Solvent -> Ramped to 50% Polar Solvent over 30 Minutes 3. 50% Polar solvent -> Maintained for 10 Minutes

4. 50% Polar Solvent -> Ramped to 80% Polar Solvent over 20 Minutes

5. 80% Polar solvent -> Maintained for 10 Minutes

Product containing fractions were combined and concentrated under high vacuum to give an almost clear & colourless wax-like oil. Yield : 0.676g, 96% Pure by LC-MS @ 254nm UPLC-MS: Rt = 1.28 min; MS m/z [M+H]⁺ 780.5; Method A.

(S)-N-((R)-1 -(4-(2,5-dif luorophenyl)-1 -(3-hydroxybenzyl)-1 H-imidazol-2-yl)-4-hvdroxy-

2.2-dimethylbutyl)-N-(g3S.4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamide

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(4-(2,5-difluorophenyl)-1- (3-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (50mg) in acetonitrile (1 ml) was added 6M HCI (0.998ml_) and the RM stirred at 60°C for 3 hours - by which time LC-MS showed that the reaction was complete with full conversion to desired. The mixture was filtered through a 0.2μιη PTFE syringe filter and the resultant clear solution was purified by reverse phase preparative-scale HPLC, Method C. Product containing fractions were combined and concentrated in vacuo to give an off-white powder. Yield : 12.9mg UPLC-MS: Rt = 0.77 min; MS m/z [M+H]⁺ 575.2; Method A. UPLC-MS: Rt = 2.87 min; MS m/z [M+H]⁺ 575.2; Method B.

(S)-N-((R)-1 -(4-(2.5-dif luorophenylH -(3-fluoro-5-hvdroxybenzyl)-1 H-imidazol-2-yl)-4- hvdroxy-2,2-dimethylbutyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamide

To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(4-(2,5-difluorophenyl)-1- (3-fluoro-5-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (50mg) in acetonitrile (1 ml) was added 6M HCI (1.03ml_) and the mixture stirred at 60°C for 2 hours - by which time LC-MS showed that the reaction was complete with full conversion to desired product The mixture was filtered through a 0.2μιη PTFE syringe filter and the resultant clear solution was purified by reverse phase preparative-scale HPLC, Method C. Product containing fractions were combined and lyophilised overnight give an off-white fluffy powder. Yield : 19.4mg UPLC-MS: Rt = 0.70 min; MS m/z [M+H]⁺ 593.1 ; Method A. UPLC-MS: Rt = 2.59 min; MS m/z [M+H]⁺ 593.2; Method B. ¹H NMR (600 MHz, DMSO- d₆) 5 10.10 (s, 1 H), 9.04 (s, 1 H), 8.71 (s, 1 H), 7.91 (s, 1 H), 7.80 - 7.73 (m, 1 H), 7.41 - 7.30 (m, 1 H), 7.18 - 7.09 (m, 1 H), 6.69 - 6.57 (m, 2H), 6.52 (dd, J = 16.2, 10.3 Hz, 1 H), 5.80 (s, 1 H), 5.39 - 5.21 (m, 2H), 4.97 (d, J = 15.5 Hz, 1 H), 4.64 - 4.57 (m, 1 H), 4.08 - 3.98 (m, 1 H), 3.95 - 3.85 (m, 1 H), 3.37 - 3.27 (m, 2H), 3.23 (dt, J = 9.9, 5.0 Hz, 2H), 2.48 - 2.36 (m, 1 H), 2.08 - 1 .95 (m, 1 H), 1.94 - 1 .86 (m, 1 H), 1 .51 (ddd, J = 23.3, 13.9, 6.7 Hz, 1 H), 1 .35 (t, J = 7.0 Hz, 3H), 1 .30 - 1 .22 (m, 1 H), 0.93 (s, 3H), 0.84 (s, 3H).

(R)-4-((S)-2-acetoxy-N-(((3R,4R)-1 -(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1 -(3-(methoxymethoxy)benzyl)- 1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid

To a cooled (0°C) solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(4-(2,5- difluorophenyl)-1 -(3-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (520mg) in anhydrous DMF (10ml_) was added pyridinium dichromate (783mg). After the addittion was complete, the dark brick red / dirty orange-coloured RM was stirred at RT overnight - by which time the colour had turned a dark brown. The RM was partioned between ethyl acetate (150ml_) and de-ionised water (100ml_).The mixture was transferred to a separating funnel and, after extraction, the aqueous phase was re-extracted with ethyl acetate (2x100ml). The combined organics were then washed with de-ionised water (100ml), sat. brine (150ml), dried over MgSC>4, filtered through No. 1 filter paper and concentrated in vacuo to give a brown wax-like oily substance. Crude yield : 563.8mg

UPLC-MS: Rt = 1 .27 min; MS m/z [M+H]⁺ 775.4; [M-H]- 773.4 Method A.

(R)-4-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-3- yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1 -(3-fluoro-5- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid

To a cooled (0°C) solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(4-(2,5- difluorophenyl)-1 -(3-fluoro-5-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4- hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (300mg) in anhydrous DMF (5mL) was added pyridinium dichromate (974mg). After the addittion was complete, the dark brick red / dirty orange-coloured RM was stirred at RT overnight - by which time the colour had turned a dark brown. The RM was partioned between ethyl acetate (100ml_) and de-ionised water (100ml_).The mixture was transferred to a separating funnel and, after extraction, the aqueous phase was re- extracted with ethyl acetate (2x100ml). The combined organics were then washed with de-ionised water (100ml), sat. brine (150ml), dried over MgSC>4, filtered through No. 1 filter paper and concentrated in vacuo to give a brown wax-like oily substance. Crude yield : 322mg UPLC-MS: Rt = 1.26 min; MS m/z [M-H]^" 791.2 Method A.

(R)-4-(4-(2.5-difluorophenyl)-1-(3-hvdroxybenzyl)-1 H-imidazol-2-yl)-4-gS)-N-

(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hvdroxypropanamido)-N,3,3- trimethylbutanamide

Step 1 : To a solution of (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1 -(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid (60mg) and DIPEA ( 189μί) in anhydrous DMF (2mL) was added HATU ( 124mg). The RM was stired at RT for 10 minutes and then methylamine hydrochloride (73mg) added. The RM was stirred at RT overnight - by which time LC-MS showed that the reaction was complete. The volatiles were removed under high vacuum and the crude residue dissolved in a 1 : 1 mixture of Ace ton itrile: Water (4ml). The RM was filtered through a 0.2μιη PTFE syringe filter and the resultant clear solution was split into 2x2ml_ batches and purified purified by reverse phase preparative-scale HPLC, Method C. Product containing fractions from both runs were combined and concentrated in vacuo to give a pale blue solid: Yield : 15mg This was taken directly to the next step:

Step 2 : BOC & MOM Deprotection: To the crude product from step 1 was added Acetonitrile ( 1 ml) and 6M HCI (1 .08ml_). The RM was stired at 50°C for 2 Hours - by which time LC-MS showed that the reaction had run to completion with deprotection to give desired final product. The RM was filtered through a 0.2μιη PTFE syringe filter and then purified by reverse phase preparative-scale HPLC, Method C. The product containing fraction was lyophilised overnight to give a white fluffy powder. Yield : 8.9mg UPLC-MS: Rt = 2.41 min; MS m/z [M+H]⁺ 603.3; Method B

1 H-NMR (HSQC) consistent with target structure.

(R)-4-(4-(2.5-difluorophenyl)-1 -(3-fluoro-5-hvdroxybenzyl)-1 H-imidazol-2-yl)-4- S)-N-

Step 1 : To a solution of (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1 -(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1 -(3-fluoro-5- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid (60mg) and DIPEA (264μί) in anhydrous DCM (2mL) was added HATU (173mg). The RM was stired at RT for 10 minutes and then methylamine hydrochloride (102mg) added. The RM was stirred at RT for 2 Hours - by which time LC-MS showed that the reaction was complete. The RM was concentrated under high vacuum to give a brown wax-like oil. This was taken forward directly to the next step.

Step 2 : BOC, MOM & Acyl Deprotection: The crude product from step 1 was dissolved in acetonitrile (2ml) and 6M HCI ( 1 .5ml_) added. The RM was stired at 60°C for 2 Hours - by which time LC-MS showed that the reaction had run to completion with deprotection to the final product. The RM was then partitioned between de-ionised water (50ml_) and ethyl acetate (50ml). After extraction, the aqueous phase was re-extracted with ethyl acetate (70ml_). The combined organics were then washed with sat. brine (70ml_), dried over MgSC>4, filtered through No.1 filter paper and concentrated in vacuo to give an off- white solid. The RM was dissolved in a 2: 1 solution of water:Acetonitrile (4ml_) and this was filtered through a 0.2μιη PTFE syringe filter. The resultant solution was split into 2x2ml_ batches and purified by reverse phase preparative-scale HPLC, Method C. The product containing fractions were combined and then lyophilised overnight to give a white fluffy powder. Yield : 18.9mg.

UPLC-MS: Rt = 2.41 min; MS m/z [M+H]⁺ 618.3; Method B

1 H-NMR (HSQC) consistent with target structure.

Synthesis of Payload— Linker Combinations

Cleavable Linkers: MC-ValCit-PABC ADCs:

To a solution of the payload (0.05 mmol, 1 eq) in DMSO (0.1 M) were added /^'Pr₂EtN (6 eq) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.09 mmol, 1 .6 eq). The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude was filtered to remove solids and directly purified by reverse phase column chromatography (using either PrepLC Method C or D) to yield the desire MC-ValCit-PABC-payload as the TFA salt.

(3R,4 )-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate.

¹H-NMR (DMSO, 600 MHz): δ 9.98 (1H, m), 8.09 (1H, m), 7.80 (1H, m), 7.74 (1H, m), 7.71 (2H, m), 7.56 (2H, m), 7.37 (3H, m), .7.31 (4H, m), 7.09 (1H, m), 7.00 (2H, m), 6.01 (1H, m), 5.68 (1H, m), 5.28 (1H, m), 5.18 (1H, m), 5.15 (2H, m), 5.01 (1H, m), 4.88 (1H, m), 4.73 (1H, m) 4.50 (1H, m), 4.39 (1H, m), 4.21 (1H, m), 3.80 (1H, m), 3.50-3.25 (8H, m), 3.37 (2H, m), 3.03 (1H, m), 2.98 (1H, m), 2.82 (1H, m), 2.60 (1H, m), 2.30 (1H, m), 2.20 (1H, m), 2.18 (1H, m), 2.14 (1H, m), 2.13 (1H, m), 2.12 (1H, m), 1.98 (1H, m), 1.71 (1H, m), 1.50 (3H, m), 1.49-1.35 (3H, m), 1.27 (3H, m), 1.23 (1H, m), 1.10 (1H, m), 0.98 (1H, m), 0.86 (6H, m), 0.70 (1H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+: 1155.5, 1.07 min (Method A).

(3R,4R)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl 3-((1-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-((S)-1- hydroxypropan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate.

¹H-NMR (DMSO, 600 MHz): δ 9.99 (1 H, m), 8.09 (1 H, m), 7.80 (1 H, m), 7.71 (2H, m), 7.57 (3H, m), 7.35 (2H, m), 7.30 (4H, m), 7.23 (1 H, m), 7.09 (1 H, m), 7.00 (2H, s), 5.97 (2H, m), 5.35 (2H, m), 5.07 (2H, m), 4.89 (1 H, m), 4.77 (1 H, m), 4.42 (1 H, m), 4.20 (1 H, m), 3.84 (1 H, m), 3.79 (1 H, m), 3.64 (2H, m), 3.40-3.23 (8H, m), 3.03 (1 H, m), 2.96 (2H, m), 2.81 (1 H, m), 2.57 (1 H, m), 2.25 (1 H, m), 2.15 (2H, m), 2.00 (1 H, m), 1.66 (2H, m), 1.52-1.25 (8H, m), 1.21 (2H, m), 1.07 (3H, m), 0.84 (6H, m), 0. 77 (1 H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1 184.5, 1 185.5 (+H), 1.04 min (Method A).

(3R,4R)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl 3-(((3S,4R)-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3,4- dihydroxypyrrolidine-1-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate.

¹H-NMR (DMSO, 600 MHz): δ 9.99 (1H, m), 8.08 (1H, m), 7.80 (1H, m), 7.71 (2H, m), 7.58 (2H, m), 7.36 (2H, m), 7.21 (2H, m), 7.20 (2H, m), 7.17 (2H, m), 7.09 (1H, m), 7.00 (2H, s), 6.01 (1H,m ), 5.75 (1H, m), 5.37 (1H, m), 5.13 (1H, m), 4.89 (3H, m), 4.41 (1H, m), 4.21 (1H, m), 4.06 (1H, m), 3.98 (1H, m), 3.82 (1H, m), 3.41 (1H, m), 3.37 (2H, m), 3.32 (2H, m), 3.13 (2H, m), 3.05 (1H, m), 2.97 (2H, m), 2.38 (1H, m), 2.35-2.05 (3H, m), 1.97 (2H, m), 1.70 (2H, m), 1.65 (2H, m), 1.60-1.40 (6H, m), 1.36 (2H, m), 1.19 (2H, m), 1.04 (1 H, m), 1.01 (1 H, m), 0.87 (6H, m), 0.46 (2H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1212.4, 2.48 min.

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl ((S)-2-(3-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)ureido)propyl)carbamate.

¹H-NMR (DMSO, 600 MHz): δ 10.03-9.92 (1H, m), 9.09-8.90 (1H, m), 8.66-8.47 (1H, m), 8.14-7.96 (1H, m), 7.84-7.70 (3H, m), 7.68-7.53 (2H, m), 7.47-7.36 (2H, m), 7.36-7.21 (7H, m), 7.17-7.06 (1H, m), 7.06-6.94 (2H, m), 6.34-6.11 (1H, m), 6.10-5.90 (1H, m), 5.41-5.09 (4H, m), 5.05-4.89 (2H, m), 4.46-4.31 (1H, m), 4.31-4.14 (1H, m), 3.91-3.79 (3H, m), 3.79- 3.69 (1H, m), 3.65-3.53 (1H, m), 3.52-3.27 (6H, m), 3.26-3.16 (1H, m), 3.16-3.07 (2H, m), 3.07-2.99 (1H, m), 2.99-2.89 (1H, m), 2.63-2.47 (2H, m), 2.45-2.29 (1H, m), 2.27-2.04 (2H, m), 2.03-1.86 (2H, m), 1.77-1.65 (1H, m), 1.65-1.55 (1H, m), 1.55-1.31 (7H, m), 1.30-1.14 (4H, m), 1.11-0.96 (3H, m), 0.91-0.78 (6H, m), 0.75-0.61 (1H, m). Missing peaks hidden under the solvent peak. LC/MS (uplc): M+ 1183.6.0.94 min (Method A).

(3R,4S)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2- hydroxypropanamido)methyl)-4-hydroxypyrrolidine-1-carboxylate

10% yield. UPLC-MS: Rt = 1.01 mins; MS m/z [M+H]⁺ 1153.4; Method A.

(R)-4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2- hydroxypropanamido)methyl)pyrrolidine-1-carboxylate

14% yield; UPLC-MS: Rt = 1.09 mins; MS m/z [M+H]⁺ 1 137.4; Method A.

Stable Linkers: a) Carbamate Attachment

To a solution of the Boc-payload (0.09 mmol, 1 eq) in DMF (0.1 M) was added /^'Pr₂EtN (0.9 mmol, 10 eq) followed by bis(4-nitrophenyl) carbonate (0.14 mmol, 1.6 eq) and stirred at room temperature for 1 h. Upon completion of the reaction by LC/MS (uplc, Method A), was added the linker (3 eq), and the reaction mixture stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), solvent was evaporated under reduce pressure and the desire product (Boc-L-P) was obtained after purification by normal phase column chromatography (PrepLC Method B).

b) Amide Attachment

To a solution of the carboxilic acid linker (0.23 mmo, 1.8 eq), HATU (0.27 mmol, 2.1 eq) in DMF(0.3 M), was added /Pr₂EtN (1.4 mmol, 1 1 eq), followed by addition of the Boc- payload (0.13 mmol, 1 eq). The reaction mixture was stirred at room tempererature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was diluted in EtOAc, and partioned between EtOAc and H₂0. The organic layer was separated and washed with H₂0 (twice), dried over Na₂S04, filtered and solvent evaporated under reduce pressure. The desire product was obtained after purification by normal phase column chromatography(PrepLC Method B). c) Click Chemistry

To a solution of azide-Boc-payload (0.05 mmol, 1 eq) and 1-(prop-2-yn-1-yl)-1 H-pyrrole- 2,5-dione (0.078 mmo, 1.5 eq) in THF (0.1 M), a solution of CuS0₄.5H₂0 and sodium ascorbate in H₂0 (1 M) was added. The reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the reaction mixture was diluted in EtOAc and partioned between EtOAc and H₂0. The organic layer was separated, washed with H₂0, dried over Na₂S0₄, filtered, and solvent evaporated under reduce pressure. The desire product was isolated by normal phase column

chromatography(PrepLC Method B). d) Sulfo GMBS coupling

Over a solution of the Boc-payload (0.05 mmol, 1 eq) in CH₂CI₂ (0.1 M) were added /Pr₂EtNH (0.30 mmol, 6 eq) and 1 -(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-4-oxo-4- (perfluorophenoxy)butane-2-sulfonic acid (0.15mmol, 3 eq). The reaction was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the solvent was evaporated under reduce pressure. The desire product was isolated as a mixture of diastero isomers by normal phase column chromatography(Prepl_C Method B).

General protocol 1 for linker attachment. i) bis(para-nitrophenol) carbonate,

DIPEA, DMF, RT, 1 h

ii) Linker rt 1 h

Payload

To a solution of the Boc-payload (1 equiv.) in dry DMF (0.1 M) was added /^'Pr₂EtN (10 equiv.) followed by bis(4-nitrophenyl) carbonate (2.2 equiv.) and the reaction was stirred at room temperature for 30 mins. Upon completion by LC/MS (uplc), the linker (4 eq) was added and the reaction stirred at room temperature for 1 h. The reaction was quenched with water and extracted with ethyl acetate. The organic extractes were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((2-(2,5-dioxo-2,5-dihydro-

1 H-pyrrol-1 -yl)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

80% yield; UPLC-MS: Rt = 1 .24 mins; MS m/z [M+H]⁺ 863.1 ; Method A.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

The product was synthesized using the general protocol 1 but employing 1 .5 equiv. of bis(4-nitrophenyl) carbonate, 1 .5 equiv. of 2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethoxy)ethanaminium chloride as the amine and 15 equiv. of DIPEA to give the desired product in 32% yield; UPLC-MS: Rt = 2.62 mins; MS m/z [M+H]⁺ 907.5; Method E. (3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propan^

1 H-pyrrol-1 -yl)hexyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

The product was synthesized using the general protocol 1 but employing 6-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1-yl)hexan-1 -aminium 2,2,2-trifluoroacetate as the amine to give the desired product in 86% yield; UPLC-MS: Rt = 1 .35 mins; MS m/z [M+H]⁺ 919.2; Method A.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-((3-((2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)methyl)azetidine-1-carbonyl)oxy)pyrrolidine-1 -carboxylate

The product was synthesized using the general protocol 1 but employing 1 .5 equiv. of bis(4-nitrophenyl) carbonate, 1 .5 equiv. of 3-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)methyl)azetidin-1 -ium 2,2,2-trifluoroacetate as the amine and 15 equiv. of DIPEA to give the desired product in 39% yield; UPLC-MS: Rt = 2.66 mins; MS m/z [M+H]⁺ 889.4; Method E.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((3-((2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)amino)-3-oxopropyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

The product was synthesized using the general protocol 1 but employing 1 .5 equiv. of bis(4-nitrophenyl) carbonate, 1.5 equiv. of 3-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethyl)amino)-3-oxopropan-1 -aminium 2,2,2-trifluoroacetate as the amine and 15 equiv. of DIPEA to give the desired product in 80% yield; UPLC-MS: Rt = 2.66 mins; MS m/z [M+H]⁺ 934.3; Method E.

(3S,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((2-(2,5-dioxo-2,5-dihydro-

1 H-pyrrol-1 -yl)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

81 % yield; UPLC-MS: Rt = 1 .27 mins; MS m/z [M+H]⁺ 863.3; Method A.

(3S,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-(((2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

50% yield; UPLC-MS: Rt = 1 .28 mins; MS m/z [M+H]⁺ 907.3; Method A.

2-yl)-2,2-dimethylpropyl)propanamido)methyl)-4-(((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

67% yield; UPLC-MS: Rt = 3.05 mins; MS m/z [M+H]⁺ 835.5; Method E.

1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-(((2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)carbamoyl)oxy)pyrrolidine-1 -carboxylate

70% yield; UPLC-MS: Rt = 1 .26 mins; MS m/z [M+H]⁺ 892.5; Method E.

(3R,4S)-tert-butyl 3-((3-((R)-1 -acetoxypropan-2-yl)-1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)ureido)methyl)-4-(((2-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamoyl)oxy)pyrrolidine-1-carboxylate

45% yield; UPLC-MS: Rt = 1.28 mins; MS m/z [M+H]⁺ 936.3; Method A.

(R)-tert-butyl 3-((S)-2-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-

2H-pyran-4-yl)methyl)-14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-methyl-3,8-dioxo-7,12- dioxa-2,4,9-triazatetradecyl)pyrrolidine-1-carboxylate

31 % yield; UPLC-MS: Rt = 1.29 mins; MS m/z [M+H]⁺ 878.2; Method A.

(3R,4R)-tert-butyl 3-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro- 2H-pyran-4-yl)methyl)-3-((S)-1-((3-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)azetidine- 1-carbonyl)oxy)propan-2-yl)ureido)methyl)-4-fluoropyrrolidine-1-carboxylate

34% yield; UPLC-MS: Rt = 1.28 mins; MS m/z [M+H]⁺ 878.2; Method A.

General protocol 2 for linker attachment

To a solution of the Boc-payload (1 equiv.) in dry DMF (0.2 M) was added /^'Pr₂EtN (3 equiv.) followed by 2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl (4-nitrophenyl) carbonate

(1.1 equiv.) and the reaction was stirred at room temperature for 1 h. Upon completion by LC/MS (uplc), the reaction was quenched with water and extracted with ethyl acetate. The organic extractes were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound. (3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-((2-

(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)carbonyl)azetidin-3-yl)methyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

42% yield; UPLC-MS: Rt = 2.49 mins; MS m/z [M+H]⁺ 795.3; Method E.

2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl 4-((R)-((S)-2-acetoxy-N-(((3R,4R)-

1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)propanamido)(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)methyl)piperidine-1-carboxylate

32% yield; UPLC-MS: Rt = 2.74 mins; MS m/z [M+H]⁺ 909.6; Method E.

General protocol 3 for linker attachment

To a solution of the carboxylic acid linker (1 .8 equiv.), HATU (2.1 equiv.) in DCM (0.2 M) were added /^'Pr₂EtN (1 1 equiv.) followed by the Boc-payload (1 equiv.) in DMF (1 :9 ratio with DCM). The reaction mixture was stirred at RT for 1 h. Upon completion of the reaction by LC/MS (uplc), the reaction mixture was diluted in EtOAc, and partitioned between EtOAc and H₂0. The organic layers were combined, dried over Na₂S0₄, filtered and solvent evaporated under reduce pressure. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(1 -(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoyl)piperidin-4- yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

99% yield; UPLC-MS: Rt = 2.58 mins; MS m/z [M+H]⁺ 849.5; Method E.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H

yl)(1 -(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)propanoyl)piperidin-4- yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

99% yield; UPLC-MS: Rt = 2.59 mins; MS m/z [M+H]⁺ 893.5; Method E.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-(3-

(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoyl)azetidin-3-yl)methyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

43% yield; UPLC-MS: Rt = 2.32 mins; MS m/z [M+H]⁺ 779.2; Method E.

General protocol 4 for linker attachment

Payload

To a solution of the Boc-payload (1.0 equiv.) and /Pr₂EtN (7.0 equiv.) in DMF (0.2 M) were added HATU (2.0 equiv.) followed by amine linker (2.0 equiv.). The reaction mixture was stirred at RT for 1 h. Upon completion of the reaction by LC/MS (uplc), the reaction mixture was diluted in EtOAc, and partitioned between EtOAc and H₂0. The organic layers were combined, dried over Na₂S0₄, filtered and solvent evaporated under reduce pressure. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-((2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)ethyl)carbamoyl)pyrrolidine-1 -carboxylate

53% yield; UPLC-MS: Rt = 2.46 mins; MS m/z [M+H]⁺ 847.0; Method E.

(3R,4S)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)propanamido)methyl)-4-((2-(2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamoyl)pyrrolidine-1 -carboxylate

27% yield; UPLC-MS: Rt = 2.48 mins; MS m/z [M+H]⁺ 890.9; Method E;

tert-butyl 3-(2-(((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((2-(2,5-dioxo-

2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4- oxobutyl)carbamoyl)phenyl)pyrrolidine-1-carboxylate

The product was synthesized using the general protocol for linker attachment 4 but employing 1.2 equiv. of HATU, 1.1 equiv of the amine linker and 5 equiv. of DIPEA to give the desired product in 89% yield; UPLC-MS: Rt = 2.47 and 2.50 mins; MS m/z [M+H]⁺ 795.4; Method E.

(R)-tert-butyl 3-(2-((1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((2-(2,5-dioxo-

2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4- oxobutyl)carbamoyl)phenoxy)azetidine-1-carboxylate

The product was synthesized using the general protocol for linker attachment 4 but employing 1 .2 equiv. of HATU, 1 .1 equiv of the amine linker and 5 equiv. of DIPEA to give the desired product in 96% yield; UPLC-MS: Rt = 2.44 mins; MS m/z [M+H]⁺ 797.3; Method E.

(3R,4S)-tert-butyl 3-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butyldimethylsilyl)oxy)propanamido)methyl)- 4-(4-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)-1 H-1 ,2,3-triazol-1 -yl)pyrrolidine-1 - carboxylate

To a solution of (3S,4R)-tert-butyl 3-azido-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2-((tert-butyldimethylsilyl)oxy) propanamido) methyl)pyrrolidine-1 -carboxylate (40 mg, 0.050 mmol) and 1 -(prop-2-yn-1 - yl)-1 H-pyrrole-2,5-dione (10 mg, 0.076 mmol) in MeCN (0.3 ml) was added a solution of copper(l) iodide (10 mg, 0.050 mmol) in water (0.3 ml) followed by triethylamine (0.07 ml, 0.050 mmol) and the reaction mixture was stirred at RT for 1 h. The reaction mixture was filtered over celite, extracted with EtOAc and washed with brine. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. Purification of the crude product by chromatography on silica eluting with 0 - 100% ethylacetate in heptane afforded the title compound as a pale yellow oil in 77% yield; UPLC-MS: Rt = 3.19 mins; MS m/z [M+H]⁺ 929.6; Method E.

General protocol 1 for simultaneous deprotection of N-BOC plus -O-acetate or -O-TBS Substrate (1 .0 equiv.) was dissolved in a 2: 1 mixture of acetonitrile and water (0.1 M). TFA (50 equiv.) was added and reaction mixture was stirred at 60°C until completion (7- 72h) as determined by UPLC-MS. The reaction was filtered and purified by reverse phase column chromatography. The product was isolated as the TFA salt as a white powder upon lyophilisation

(3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)carbamate

41 % yield; UPLC-MS: Rt = 0.86 mins; MS m/z [M+H]⁺ 721 .1 ; Method A. ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 9.1 1 -8.63 (2H, m), 7.91 -7.51 (2H, m), 7.45-7.05 (8H, m), 7.04-6.92 (2H, m), 5.65-4.98 (3H, m), 4.94-4.82 (1 H, m), 4.55-4.44 (1 H, m), 3.89-2.84 (13H, m), 2.69-1 .70 (4H, m), 1 .55-0.48 (7H, m).

(3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate

24% yield; UPLC-MS: Rt = 0.89 mins; MS m/z [M+H]⁺ 765.1 ; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 9.07-8.71 (2H, m), 7.89-7.73 (2H, m), 7.42-7.07 (8H, m), 7.02-6.99 (2H, m), 5.65-5.49 (1 H, m), 5.40-5.03 (2H, m), 4.49-4.47 (1 H, m), 3.92-2.91 (18H, m), 2.69-2.10 (3H, m), 1 .53-0.52 (7H, m).

(3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (6-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)hexyl)carbamate

37% yield; UPLC-MS: Rt = 0.95 mins; MS m/z [M+H]⁺ 777.2; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.96-8.75 (2H, m), 7.88-7.54 (2H, m), 7.43-7.26 (6H, m), 7.17-7.07 (2H, m), 7.01 -7.00 (2H, m), 5.65-5.04 (3H, m), 4.94-4.85 (1 H, m), 4.50-4.42 ( 1 H, m), 3.94-2.83 (14H, m), 2.68-1 .75 (3H, m), 1 .52-0.51 ( 15H, m).

(3S,4S)-4-(((S)-N-((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl 3-((2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)methyl)azetidine-1 -carboxylate

40% yield; UPLC-MS: Rt = 0.90 mins; MS m/z [M+H]⁺ 747.2; Method A; ¹ H-NMR (CDCI₃, 400 MHz, mixture of rotamers): δ 10.25-9.50 (2H, m), 7.93-7.56 (2H, m), 7.46-7.27 (5H, m), 7.22-6.91 (2H, m), 6.76-6.74 (2H, m), 5.98-4.44 (3H, m), 4.05-3.58 (10H, m), 3.45- 3.18 (3H, m), 3.09-2.19 (9H, m), 1 .40-0.64 (7H, m).

(3S,4S)-4-(((S)-N-((R)-( 1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (3-((2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)amino)-3-oxopropyl)carbamate

26% yield; UPLC-MS: Rt = 0.84 mins; MS m/z [M+H]⁺ 792.1 ; Method A; ¹ H-NMR (CDCI₃, 400 MHz, mixture of rotamers): δ 9.97-9.51 (2H, m), 7.88-7.61 (2H, m), 7.44-7.29 (5H, m), 7.14-6.81 (2H, m), 6.73-6.69 (2H, m), 5.98-4.44 (5H, m), 3.97-3.94 (1 H, m), 3.75-2.32 (22H, m), 1 .43-0.66 (7H, m).

(3R,4R)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)carbamate

30% yield; UPLC-MS: Rt = 0.87 mins; MS m/z [M+H]⁺ 720.3; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.96-8.85 (2H, m), 7.92-7.66 (2H, m), 7.42-6.89 (10H, m), 5.66-5.03 (3H, m), 4.54-4.43 (1 H, m), 4.08-2.93 (18H, m), 1 .52-0.51 (7H, m). (3S,4S)-4-(((S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylpropyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)ethyl)carbamate

(3R,4R)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate

42% yield; UPLC-MS: Rt = 0.89 mins; MS m/z [M+H]⁺ 765.2; Method A; ¹ H-NMR (DMSO, 600 MHz, mixture of rotamers): δ 8.97-8.85 (2H, m), 7.93-7.66 (2H, m), 7.42-6.84 (10H, m), 5.67-5.04 (3H, m), 4.55-4.43 (1 H, m), 4.09-2.93 (21 H, m), 1 .52-0.51 (7H, m).

(3S,4S)-4-(((S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2,2- dimethylpropyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)ethyl)carbamate

60% yield; UPLC-MS: Rt = 2.05 mins; MS m/z [M+H]⁺ 693.2; Method E;

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(3-(2,5-dioxo-2,5-dihydro-

1H-pyrrol-1-yl)propanoyl)piperidin-4-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)meth

2-hydroxypropanamide

30% yield; UPLC-MS: Rt = 1.57 mins; MS m/z [M+H]⁺ 707.4; Method E.¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.98-8.90 (1H, m), 8.64-8.56 (1H, m), 7.78-7.61 (2H, m), 7.34-7.18 (6H, m), 7.06-7.02 (1H, m), 6.93 (2H, s), 5.60-5.44 (1H, m), 5.34-4.96 (3H, m), 4.46-4.29 (1H, m), 4.19-1.97 (17H, m), 1.42-0.29 (7H, m).

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(1-(3-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)propanoyl)piperidin-4-yl)methyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

29% yield; UPLC-MS: Rt = 1.59 mins; MS m/z [M+H]⁺ 751.5; Method E. ¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.02-8.93 (1 H, m), 8.66-8.57 (1 H, m), 7.85-7.69 (2H, m), 7.42-7.24 (6H, m), 7.15-7.07 (1 H, m), 7.01 (2H, s), 5.67-5.51 (1 H, m), 5.42-5.03 (3H, m), 4.53-4.35 (1 H, m), 4.27-2.05 (22H, m), 1.51-0.34 (7H, m).

2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl 4-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamido)methyl)piperidine-1-carboxylate

21 % yield; UPLC-MS: Rt = 1.70 mins; MS m/z [M+H]⁺ 767.5; Method E.

(3S,4S)-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-N-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-

1-yl)ethyl)pyrrolidine-3-carboxamide

28% yield; UPLC-MS: Rt = 1.77 mins; MS m/z [M+H]⁺ 705.3; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.64-8.51 (2H, m), 8.34-8.32 (1H, m), 7.82-7.67 (2H, m), 7.43-7.27 (6H, m), 7.15-7.09 (1H, m), 7.02-7.00 (2H, m), 5.62-4.47 (1H, m), 5.36-5.01 (2H, m), 4.51-4.39 (1H, m), 3.89-2.99 (18H, m), 1.83-0.50 (7H, m).

(3S,4S)-4-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H- pyran-4-yl)methyl)-2-hydroxypropanamido)methyl)-N-(2-(2-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)ethoxy)ethyl)pyrrolidine-3-carboxamide

75% yield; UPLC-MS: Rt = 1.80 mins; MS m/z [M+H]⁺ 749.4; Method E; ¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 8.62-8.58 (2H, m), 8.32-8.27 (1H, m), 7.83-7.64 (2H, m), 7.43-7.27 (6H, m), 7.15-7.09 (1H, m), 7.04-7.02 (2H, m), 5.63-4.46 (1H, m), 5.36-5.03 (2H, m), 4.44-4.40 (1H, m), 3.88-3.08 (21 H, m), 2.84-2.62 (1H, m), 1.91-0.51 (7H, m).

(3S,4S)-4-((1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran- 4-yl)methyl)-3-((S)-1-hydroxypropan-2-yl)ureido)methyl)pyrrolidin-3-yl (2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethyl)carbamate

29% yield; UPLC-MS: Rt = 0.87 mins; MS m/z [M+H]⁺ 750.2; Method A;

(3S^S)-4-((1 -((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran- 4-yl)methyl)-3-((S)-1 -hydroxypropan-2-yl)ureido)methyl)pyrrolidin-3-yl (2-(2-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate

48% yield; UPLC-MS: Rt = 0.88 mins; MS m/z [M+H]⁺ 794.2; Method A;

General protocol 2 for overall deprotection

Substrate (1 .0 equiv.) was dissolved DCM (0.1 M) and TFA (20-50 equiv.) was added and reaction mixture was stirred at RT for 1 -2 h. The reaction was filtered and purified by reverse phase column chromatography. The product was isolated as the TFA salt as a white powder upon lyophilisation

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-N-(((3S,4S)-4-(4-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)methyl)-1 H-1 ,2,3- triazol-1-yl)pyrrolidin-3-yl)methyl)-2-hydroxypropanamide

50 equiv. TFA, 24% yield; UPLC-MS: Rt = 1.65 mins; MS m/z [M+H]⁺ 715.4; Method E. ¹ H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.04-8.82 (2H, m), 8.09-8.04 (1 H, m), 7.88-7.76 (1 H, m), 7.71 -7.60 (1 H, m), 7.42-7.05 (9H, m), 5.58-5.55 (1 H, m), 5.39-4.98 (5H, m), 4.68-4.66 (2H, m), 4.01- 2.46 (10H, m), 1.47-0.45 (9H, m).

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1-(3-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1-yl)propanoyl)azetidin-3-yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)- 2-hydroxypropanamide

50 equiv. TFA; 26% yield; UPLC-MS: Rt = 1.46 mins; MS m/z [M+H]⁺ 679.2; Method E. ¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.04 (1H, s, br), 8.69 (1H, s, br), 7.83- 7.65 (2H, m), 7.43-7.29 (6H, m), 7.15-7.10 (1H, m), 7.02-6.97 (2H, m), 6.25-5.65 (1H, m), 5.52-5.07 (3H, m), 4.56-4.26 (1H, m), 4.16-2.76 (17H, m), 2.48-2.08 (3H, m), 1.30-0.97 (3H, m).

2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl 3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H- imidazol-2-yl)((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamido)methyl)azetidine-1-carboxylate

50 equiv. TFA; 60% yield; UPLC-MS: Rt = 1.65 mins; MS m/z [M+H]⁺ 695.3; Method E. ¹H-NMR (DMSO, 400 MHz, mixture of rotamers): δ 9.05 (1H, s, br), 8.69 (1H, s, br), 7.84- 7.66 (2H, m), 7.43-7.29 (6H, m), 7.15-7.10 (1H, m), 7.02-6.98 (2H, m), 6.25-5.70 (1H, m), 5.51-5.08 (3H, m), 4.51-4.26 (1H, m), 4.15-2.76 (17H, m), 2.43-2.07 (3H, m), 1.29-0.97 (3H, m).

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl 3-((2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)methyl)azetidine-1-carboxylate

50 equiv. TFA; 75% yield; UPLC-MS: Rt = 1.78 mins; MS m/z [M+H]⁺ 778.3; Method E;

N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((2-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)-2-(pyrrolidin-3-yl)benzamide

20 equiv. of TFA; mixture of diastereoisomers; 88% yield; UPLC-MS: Rt = 1.72 mins; MS m/z [M+H]⁺ 695.2; Method E; ¹H-NMR (DMSO, 400 MHz): δ 9.50-9.46 (1H, m), 8.87-8.82 (2H, m), 8.12-8.10 (1H, m), 7.80-7.73 (1H, m), 7.53-7.26 (11H, m), 7.10-7.06 (1H, m), 6.97 (2H, s), 5.54-5.38 (3H, m), 3.76-3.57 (1H, m), 3.54-2.89 (8H, m), 2.87-2.80 (1H, m), 2.30-1.82 (3H, m), 1.10-1.08 (3H, m), 0.86-0.85 (3H, m). (R)-2-(azetidin-3-yloxy)-N-(1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)benzamide

20 equiv. of TFA; 78% yield; UPLC-MS: Rt = 1.70 mins; MS m/z [M+H]⁺ 697.2; Method E; ¹H-NMR (DMSO, 400 MHz): δ 9.02-9.00 (1 H, m), 8.87 (2H, s, br), 8.09-8.06 (1 H, m), 7.79-7.75 (1 H, m), 7.64-7.62 (1 H, m), 7.59-7.58 (1 H, m), 7.47-7.42 (1 H, m), 7.36-7.26 (6H, m), 7.12-7.06 (2H, m), 6.85 (2H, s), 6.82-6.80 (1 H, m), 5.53-5.38 (3H, m), 5.18-5.12 (1 H, m), 4.52-4.45 (1 H, m), 4.40-4.36 (1 H, m), 4.28-4.22 (1 H, m), 4.18-4.12 (1 H, m), 3.46-3.42 (2H, m), 3.35-3.27 (1 H, m), 3.19-3.1 1 (1 H, m), 2.74-2.70 (1 H, m), 2.20-2.17 (1 H, m), 1.03 (3H, s), 0.89 (3H, s).

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-((S)-pyrrolidin-3-ylmethyl)ureido)propyl (2-(2-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl)ethoxy)ethyl)carbamate

40% yield; UPLC-MS: Rt = 1.72 mins; MS m/z [M+H]⁺ 778.4; Method E. General Procedure for Boc-deprotection of Linker-Payloads:

To the above Boc-L-P combination (0.008 mmol) in MeCN (0.1 M), was added trifluoroacetic acid (0.5 ml_, 6.5 mmol), and the reaction mixture was stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude was purified by reverse phase column chromatography (using either PrepLC Method C or D). The product is isolated as the TFA salt.

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-4-(((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)(2- hydroxyethyl)carbamoyl)oxy)-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl using (S)-tert-butyl 2-amino-3- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoate (Linker 3, TFA salt) (45 mg, 0.127 mmol).

The desired product was used in the next step without further purification. LC/MS (Method A): [M+H]⁺ 967.5, Rt 1 .42 min. (S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 896.6, 1.31 min (Method A).

5 mg, 0.005 mmol, 67%. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, m), 8.67 (1 H, m), 7.77 (3H, m), 7.39 (2H, m), 7.32 (2H, m), 7.27 (2H, m), 7.10 (1 H, m), 7.01 (2H, m), 6.33 (1 H, m), 5.34 (2H, m), 5.25 (1 H, m), 5.20 (1 H, m), 3.96 (2H, m), 3.89 (1 H, m), 3.83 (1 H, m), 3.73 (1 H, m), 3.60 (1 H, m), 3.56 (4H, m), 3.39 (2H, m), 3.35 (1 H, m), 3.30 (1 H, m), 3.09 (2H, m), 2.59 (1 H, m), 2.54 (1 H, m), 2.26 (1 H, m), 2.00 (1 H, m), 1.45 (1 H, m), 1.25-1.05 (2H, m), 1.10 (3H, m), 0.75 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 796.8, 0.92 min (Method A).

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (6-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)hexyl)carbamate.

Boc-linker-payload: LC/MS (uplc): M+ 908.6, 1.37 min (Method A).

4.5 mg, 0.005 mmol, 53%. ¹H-NMR (DMSO, 600 MHz): δ 9.02 (1 H, m), 8.65 (1 H, m), 7.77 (2H, m), 7.39 (2H, m), 7.32 (2H, m), 7.27 (2H, m), 7.10 (1 H, m), 7.01 (1 H, m), 6.99 (2H, m), 6.32 (1 H, m), 5.33 (2H, m), 5.24 (1 H, m) 5.20 (1 H, m), 3.94 (1 H, m), 3.83 (1 H, m), 3.72 (1 H, m), 3. 60 (1 H, m), 3.38 (3H, m), 3.32 (3H, m), 3.21 (1 H, m), 2.95 (2H, m), 2.60 (1 H, m), 2.53 (1 H, m), 2.27 (1 H, m), 2.00 (1 H, m), 1.47 (2H, m), 1.36 (2H, m), 1.21 (5H, m), 1.09 (4H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 808.5, 1.0 min (Method A).

N-((R)-(l-benzyl-4-(2,5-difluorophenyl)-lH-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-iodoacetamide.

Boc-Linker-Payload: LC/MS (uplc): MH+ 753.3, 1.40 min (Method A).

Linker-Payload: LC/MS (uplc): MH+ 653.2, 0.94 min (Method A). (S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2- iodoacetamido)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): M+ 984.3, 1.27 min (Method A). Linker-payload: LC/MS (uplc): MH+ 884.4, 0.91 min (Method A).

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (6-(2- iodoacetamido)hexyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 996.6, 1.33 min (Method A).

4 mg, 0.004 mmol, 40%. ¹H-NMR (DMSO, 600 MHz): δ 9.00 (1 H, m), 8.63 (1 H, m), 8.19 (1 H, m), 7.77 (3H, m), 7.39 (2H, m), 7.30 (1 H, m), 7.27 (2H, m), 7.10 (1 H, m), 7.05 (1 H, m), 6.31 (1 H, m), 5.33 (2H, m) 5.23 (1 H, m), 5.19 (1 H, m), 3.94 (4H, m), 3.83 (1 H, m), 3.71 (1H, m), 3.60 (2H, m), 3.47 (1H, m), 3.34 (3H, m), 3.20 (1H, m), 3.02 (2H, m), 2.95 (2H, m), 2.58 (1H, m), 2.54 (1H, m), 2.25 (1H, m), 2.00 (1H, m), 1.37 (5H, m), 1.24 (5H, m), 1.08 (3H, m), 0.69 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 896.5, 0.97 min (Method A).

1-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-((S)-1-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-1,2,3-triazol-1- yl)propan-2-yl)-1-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)urea.

Boc-linker-payload: LC/MS (uplc): MH+ 846.5, 1.31 min (Method A).

5.5 mg, 0.006 mmol, 86%.¹H-NMR (DMSO, 600 MHz): δ 9.07 (1H, m), 8.52 (1H, m), 8.03 (1H, m), 7.79 (1H, m), 7.75 (1H, m), 7.38 (2H, m), 7.32 (2H, m), 7.24 (2H, m), 7.11 (1H, m), 7.09 (2H, m), 6.42 (1H, m), 5.30-5.15 (4H, m), 4.67 (2H, m), 4.42 (2H, m), 4.21 (1H, m), 3.81 (1H, m), 3.58 (1H, m), 3.39 (3H, m), 3.30 (1H, m ), 3.20 (1H, m), 2.53 (1H, m), 2.47 (1H, m), 2.30 (1H, m), 1.90 (1H, m), 1.35 (1H, m), 1.25 (1H, m), 1.11 (4H, m), 0.65 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 746.2, 0.92 min (Method A).

(S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 867.4, 1.33 min (Method A).

2 mg, 0.002 mmol, 36%.¹H-NMR (DMSO, 600 MHz): δ 9.11 (1H, m), 8.80 (1H, m), 7.78 (2H, m), 7.51 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.22 (2H, m), 7.10 (1H, m), 7.02 (2H, m), 5.61 (1H, m), 5.52 (1H, m), 5.23 (1H, m), 5.09 (1H, m), 5.02 (1H, m), 3.83 (1H, m), 3.75 (1H, m), 3.62 (1H, m), 3.54 (4H, m), 3.30 (1H, m), 3.23 (2H, m), 3.09 (2H, m), 2.66 (1H, m), 2.61 (1H, m), 2.31 (1H, m), 2.18 (1H, m), 1.42 (3H, m), 1.38 (2H, m), 1.10 (1H, m), 0.67 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 767.3, 0.92 min (Method A).

(S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-1-oxopropan-2-yl (6-(2,5-dioxo- 2,5-dihydro-1H-pyrrol-1-yl)hexyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 879.6, 1.40 min (Method A). 3.2 mg, 0.0034 mmol, 27%.¹H-NMR (DMSO, 600 MHz): δ 9.20-9.06 (1H, m), 8.97-8.33 (1H, m), 7.83-7.73 (2H, m), 7.56-7.46 (1H, m), 7.44-7.36 (2H, m), 7.35-7.28 (2H, m), 7.27- 7.19 (2H, m), 7.17-7.08 (1H, m), 7.03-6.96 (2H, m), 5.66-5.59 (1H, m), 5.59-5.43 (1H, m), 5.26-4.98 (3H, m), 3.88-3.77 (1H, m), 3.77-3.68 (1H, m), 3.36-3.27 (4H, m), 3.27-3.17 (2H, m), 3.07-2.96 (1H, m), 2.95-2.82 (1H, m), 2.72-2.56 (2H, m), 2.37-2.24 (1H, m), 2.23-2.05 (1H, m), 1.52-1.28 (9H, m), 1.28-1.16 (2H, m), 1.16-0.97 (3H, m), 0.73-0.56 (1H, m).

Missing signal hidden under the solvent peak. LC/MS (uplc): MH+ 779.4, 1.01 min (Method A).

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): M+ 940.4, 1.29 min (Method A).

4 mg, 0.004 mmol, 47%.¹H-NMR (DMSO, 600 MHz): δ 8.95 (1H, m), 8.45 (1H, m), 7.79 (1H, m), 7.76 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.11 (1H, m), 7.06 (1H, m), 7.03 (2H, m), 6.32 (1H, m), 5.34 (2H, m), 5.15-5.25 (2H, m), 3.96 (1H, m), 3.91 (1H, m), 3.85 (1H, m), 3.75 (1H, m), 3.60 (1H, m), 3.55-3.25 (12H, m), 3.18 (1H, m), 3.11 (2H, m), 2.65 (2H, m), 2.51 (1H, m), 2.28 (1H, m), 2.00 (1H, m), 1.40 (2H, m), 1.23 (1H, m), 1.10 (3H, m), 0.65 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 840.3, 0.94 min (Method A). (S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 852.4, 1.28 min (Method A).

10 mg, 0.011 mmol, 69%.¹H-NMR (DMSO, 600 MHz): δ 9.03 (1H, m), 8.64 (1H, m), 7.78 (1H, m), 7.77 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.20 (1H, m), 7.08 (1H, m), 7.01 (2H, m), 6.92 (1H, m), 6.30 (1H, m), 5.34 (2H, m), 5.22 (2H, m), 3.90 (3H, m), 3.80 (1H, m), 3.74 (1H, m), 3.60 (1H, m), 3.33 (2H, m), 3.20 (1H, m), 3.13 (2H, m), 2.61 (1H, m), 2.54 (1H, bs), 2.28 (1H, m), 1.99 (1H, m), 1.45 (1H, m), 1.30-1.15 (2H, m), 1.11 (3H, m), 0.68 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 752.3, 0.90 min (Method A).

2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)ethyl (2-(2-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-Linker-Payload: LC/MS (uplc): MH+ 882.2, 1.26 min (Method A).

14 mg, 0.015 mmol, 71 %. ¹H-NMR (DMSO, 600 MHz): δ 9.1 1-9.00 (1 H, m), 8.75-8.60 (1 H, m), 7.81-7.71 (2H, m), 7.43-7.36 (2H, m), 7.36-7.24 (4H, m), 7.14-7.06 (1 H, m), 7.05- 7.00 (2H, m), 7.00-6.95 (1 H, m), 6.79-6.65 (1 H, m), 5.38-5.1 1 (4H, m), 4.02-3.95 (2H, m), 3.71-3.64 (1 H, m), 3.64-3.59 (1 H. m), 3.59-3.54 (2H, m), 3.54-.344 (3H, m), 3.44-3.26 (7H, m), 3.26-3.16 (1 H, m), 3.13-3.04 (2H. m), 2.65-2.44 (2H, m), 2.43-2.26 (1 H, m), 2.02-1.83 (1 H, m), 1.45-1.33 (1 H, m), 1.30-1.12 (2H, m), 0.78-0.60 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 782.2, 0.90 min (Method A).

N-((S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-3-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)propanamide.

Boc-linker-payload: LC/MS (uplc): MH+ 880.4, 1.25 min (Method A).

10.5 mg, 0.011 mmol, 89 %.¹H-NMR (DMSO, 600 MHz): δ 9.06 (1H, m), 8.70 (1H, m), 7.99 (1H, m), 7.78 (1H, m), 7.76 (1H, m), 7.40 (2H, m), 7.33 (2H, m), 7.28 (2H, m), 7.10 (1H, m), 7.02 (2H, m), 6.35 (1H, m), 5.40-5.10 (4H, m), 3.83 (3H, m), 3.72 (1H, m), 3.45 (2H, m), 3.37-3.25 (4H, m), 3.25-3.05 (4H, m), 2.57 (1H, m), 2.50 (2H, m), 2.42 (1H, m), 2.30 (2H, m), 1.92 (1H, m), 1.40 (1H, m), 1.25 (1H, m), 1.22 (1H, m), 1.04 (3H, m), 0.70 (1H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 780.3, 0.93 min (Method A).

2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl ((S)-2-(3-((R)-(1-benzyl-4-(2,5- difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)ureido)propyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 896.2, 1.27 min (Method A).

9 mg, 0.009 mmol, 40%. ¹H-NMR (DMSO, 600 MHz): δ 9.1 1-8.96 (1 H, m), 8.74-8.59 (1 H, m), 7.82-7.69 (2H, m), 7.45-7.36 (2H, m), 7.35-7.20 (5H, m), 7.15-7.06 (1 H, m), 7.05-6.98 (2H, m), 6.34-6.15 (1 H, m), 5.38-5.12 (4H, m), 4.08-3.96 (2H, m), 3.90-3.79 (2H, m), 3.79- 3.67 (1 H, m), 3.40-3.27 (4H, m), 3.26-3.18 (1 H, m), 3.12-2.98 (2H, m), 2.65-2.46 (3H, m), 2.46-2.30 (1 H, m), 2.04-1.87 (1 H, m), 1.46-1.34 (1 H, m), 1.31 -1.12 (2H, m), 1.09-0.97 (3H, m), 0.78-0.60 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 796.2, 0.93 min (Method A).

N-((S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-6-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)hexanamide.

Boc-linker-payload: LC/MS (uplc): MH+ 878.3, 1.28 min (Method A).

19 mg, 0.02 mmol, 31 %. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, m), 8.72 (1 H, m), 7.97 (1 H, m), 7.77 (2H, m), 7.40 (2H, m), 7.35 (2H, m), 7.29 (2H, m), 7.1 1 (1 H, m), 7.01 (2H, m), 6.40 (1 H, m), 5.33 (2H, m), 5.28-5.20 (2H, m), 3.84 (2H, m), 3.70 (1 H, m), 3.60 (1 H, m), 3.45 (1 H, m), 3.37 (2H, m), 3.33 (2H, m), 3.22 (2H, m), 3.10 (1 H, m), 2.57 (2H, m), 2.51 (2H, m), 2.44 (1 H, m), .2.07 (2H, m), 1.92 (1 H, m), 1.49 (3H, m), 1.38 (1 H, m), 1.24 (1 H, m), .1.19 (2H, m), 1.04 (3H, m), 0.70 (1 H, m). LC/MS (uplc): MH+778.2, 0.96 min (Method A). (3S,4R)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2^

yl)methyl)(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-y^ (2- (2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate and

(3R^S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-yl (2- (2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

The 2 different isomers were prepared by sequential protection-deprotection steps of the 2 OH to generate the corresponding Linker-positional isomer as pure isomer each.

Structure determination to assign each postional isomer was not done.

Isomer A: Boc-linker-payload: LC/MS (uplc): M+ 1038.7, 1.50 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): MH+ 1039.2, 1.49 min (Method A).

Isomer A: 2.3 mg, 0.002 mmol, 30%. ¹H-NMR (DMSO, 600 MHz): δ 9.04 (1 H, m), 8.74 (1 H, m), 7.76 (2H, m), 7.39 (2H, m), 7.33 (2H, m), 7.25 (2H, m), 7.09 (2H, m), 7.02 (2H, m), 5.67 (1 H, m), 5.38 (1 H, m), 5.25 (1 H, m), 4.93 (1 H, m), 4.71 (1 H, m), 4.27 (1 H, m), 3.80 (1 H, m), 3.71 (2H, m), 3.57 (4H, m), 3.40 (2H, m), 3.30 (4H, m), 3.27 (4H, m), 3.1 1 (2H, m), 2.81 (1 H, m), 2.37 (1 H, m), 2.22 (1 H, m), 2.17 (1 H, m), 1.70 (1 H, m), 1.20 (1 H, m), 1.05 (1 H, m), 0.68 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 824.1 , 0.90 min (Method A).

Isomer B: 6.2 mg, 0.006 mmol, 43%. ¹H-NMR (DMSO, 600 MHz): δ 9.03 (1 H, m), 8.76 (1 H, m), 7.75 (2H, m), 7.39 (2H, m), 7.33 (2H, m), 7.25 (2H, m), 7.10 (2H, m), 7.03 (2H, m), 5.71 (1 H, m), 5.37 (1 H, m), 5.25 (1 H, m), 4.89 (1 H, m), 4.77 (1 H, m), 4.24 (1 H, m), 3.83 (1 H, m), 3.78 (1 H, m), 3.58 (4H, m), 3.51 (3H, m), 3.40 (2H, m), 3.29 (1 H, m), 3.14 (2H, m), 3.10 (2H, m), 2.80 (1 H, m), 2.36 (2H, m), 2.30 (1 H, m), 2.13 (1 H, m), 1.72 (1 H, m), 1.08 (1 H, m), 0.88 (1 H, m), 0.33 (1 H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 824.1 , 0.91 min (Method A).

(3R,4S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-yl (2- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)carbamate and

(3S,4R)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-yl (2- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)carbamate.

Structure determination to assign each postional isomer was not done. Isomer A: Boc-linker-payload: LC/MS (uplc): MH+ 995.2, 1.52 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): M+ 994.2, 1.51 min (Method A).

Isomer A: 11 mg, 0.012 mmol, 58 %.¹H-NMR (DMSO, 600 MHz): δ 9.09-8.94 (1 H, m), 8.71-8.53 (1H, m), 7.82-7.69 (2H, m), 7.42-7.36 (2H, m), 7.36-7.21 (5H, m), 7.16-7.06 (1H, m), 7.03-6.97 (2H, m), 5.72-5.62 (1H, m), 5.44-5.34 (1H, m), 5.34-5.16 (1H, m), 4.94-4.86 (1H, m), 4.74-4.66 (1H, m), 4.33-4.21 (1H, m), 3.88-3.76 (1H, m), 3.76-3.65 (2H, m), 3.65- 3.53 (1H, m), 3.22-3.16 (4H, m), 3.16-3.03 (2H, m), 2.90-2.77 (1H, m), 2.45-2.10 (3H, m), 1.77-1.67 (1H, m), 1.12-0.99 (1H, m), 0.91-0.81 (1H, m), 0.34-0.15 (1H, m). 1H, m), 4.74-4.66 (1H, m), 4.33-4.21 (1H, m), 3.88-3.76 (1H, m), 3.76-3.65 (2H, m), 3.65-3.53 (1H, m), 3.22-3.16

Isomer B: 10 mg, 0.0011 mmol, 94 %, ¹H-NMR (DMSO, 600 MHz): δ 9.11-8.94 (1H, m), 8.80-8.57 (1H, m), 7.84-7.72 (2H, m), 7.44-7.36 (2H, m), 7.36-7.28 (3H, m), 7.28-7.20 (2H, m), 7.14-7.07 (1H, m), 7.05-6.99 (2H, m), 5.79-5.69 (1H, m), 5.43-5.33 (1H, m), 5.31-5.16 (1H, m), 4.91-4.82 (1H, m), 4.82-4.72 (1H, m), 4.27-4.17 (1H, m), 3.92-3.74 (2H, m), 3.24- 3.02 (4H, m), 2.83-2.71 (1H, m), 2.44-2.25 (3H, m), 2.14-2.03 (1H, m), 1.76-1.64 (1H, m), 1.12-0.98 (1H, m), 0.92-0.79 (1H, m), 0.37-0.19 (1H, m). 9.11-8.94 (1H, m), 8.80-8.57 (1H, m), 7.84-7.72 (2H, m), 7.44-7.36 (2H, m), 7.36-7.28 (3H, m), 7.28-7.20 (2H, m),

(3R,4S)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-yl (6- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexyl)carbamate and

(3S,4R)-1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)-4-hydroxypyrrolidin-3-yl (6- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexyl)carbamate.

Structure determination to assign each postional isomer was not done.

Isomer A: Boc-linker-payload: LC/MS (uplc): M+ 1050.3, 1.57 min (Method A).

Isomer B: Boc-linker-payload: LC/MS (uplc): M+ 1050.3, 1.57 min (Method A). Isomer A: 12 mg, 0.012 mmol, 83 %. ¹H-NMR (DMSO, 600 MHz): δ 9.06-8.93 (1 H, m), 8.74-8.61 (1 H, m), 7.84-7.69 (2H, m), 7.45-7.28 (4H, m), 7.28-7.21 (2H, m), 7.18-7.06 (2H, m), 7.05- 6.96 (2H, m), 5.70-5.59 (1 H, m), 5.43-5.33 (1 H, m), 5.33-5.17 (1 H, m), 4.99-4.88 (1 H, m),

4.75- 4.63 (1 H, m), 4.33-4.20 (1 H, m), 3.85-3.75 (1 H, m), 3.75-3.63 (2H, m), 3.63-3.52 (1 H, m), 3.30-3.19 (3H, m), 3.17-3.05 (1 H, m), 3.01-2.89 (2H, m), 2.87-2.74 (1 H, m), 2.44-2.08 (3H, m), 1.77-1.62 (1 H, m), 1.54-1.44 (2H, m), 1.43-1.32 (2H, m), 1.31-1.13 (4H, m), 1.09- 0.95 (1 H, m), 0.93-0.79 (1 H, m), 0.31-0.15 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 836.2, 0.95 min (Method A), 4.00 min (Method F).

Isomer B: 9.5 mg, 0.0095 mmol, 72 %. ¹H-NMR (DMSO, 600 MHz): δ 9.07-8.93 (1 H, m),

8.76- 8.63 (1 H, m), 7.80-7.70 (2H, m), 7.43-7.36 (2H, m), 7.36-7.28 (2H, m), 7.27-7.21 (2H, m), 7.18-7.13 (1 H, m), 7.13-7.06 (1 H, m), 7.04-6.98 (2H, m), 5.80-5.63 (1 H, m), 5.42-5.32 (1 H, m), 5.32-5.12 (1 H, m), 4.97-4.83 (1 H, m), 4.82-4.72 (1 H, m), 4.23-4.17 (1 H, m), 3.89- 3.72 (2H, m), 3.72-3.56 (2H, m), 3.31-3.24 (2H, m), 3.21-3.05 (2H, m), 3.03-2.88 (2H, m), 2.86-2.71 (1 H, m), 2.44-2.23 (2H, m), 2.21-1.99 (1 H, m), 1.80-1.65 (1 H, m), 1.55-1.43 (2H, m), 1.43-1.32 (2H, m), 1.31-1.14 (4H, m), 1.13-0.98 (1H, m), 0.94-0.80 (1H, m), 0.40-0.33 (1H, m). Missing signals hidden under the solvent peak.

(R)-1-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)-3-hydroxypropan-2-yl (2-(2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): M+ 1026.3, 1.51 min (Method A), 7.10 min (Method B).

6.1 mg, 0.006 mmol, 51%.¹H-NMR (DMSO, 600 MHz): δ 9.15-8.96 (1H, m), 8.69-8.48 (1H, m), 7.83-7.67 (2H, m), 7.47-7.37 (2H, m), 7.37-7.21 (4H, m), 7.18-7.07 (1H, m), 7.07- 6.99 (2H, m), 6.98-6.86 (1H, m), 6.80-6.59 (1H, m), 5.38-5.09 (4H, m), 4.78-4.63 (1H, m), 3.88-3.77 (1H, m), 3.54-3.43 (4H, m), 3.42-3.26 (6H, m), 3.25-3.14 (2H, m), 3.15-2.98 (2H, m), 2.61-2.44 (3H, m), 2.43-2.25 (2H, m), 2.00-1.78 (1H, m), 1.43-1.30 (1H, m), 1.30-1.08 (2H, m), 0.76-0.57 (1H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 812.2, 0.86 min (Method A), 3.37 min (Method B).

(R)-3-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)-2-hydroxypropyl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): M+ 1026.3, 1.52 min (Method A), 7.17 min (Method B).

4.0 mg, 0.004 mmol, 70%. ¹H-NMR (DMSO, 600 MHz): δ 9.09-8.93 (1 H, m), 8.59-8.37 (1 H, m), 7.85-7.70 (2H, m), 7.45-7.36 (2H, m), 7.36-7.23 (4H, m), 7.15-7.06 (2H, m), 7.06-

7.01 (2H, m), 6.68-6.56 (1 H, m), 5.40-5.12 (4H, m), 4.06-3.95 (1 H, m), 3.90-3.77 (2H, m), 3.77-3.65 (2H, m), 3.26-2.97 (5H, m), 2.64-2.46 (3H, m), 2.46-2.27 (1 H, m), 1.98-1.81 (1 H, m), 1.45-1.36 (1 H, m), 1.30-1.10 (2H, m), 0.77-0.60 (1 H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 812.2, 0.86 min (Method A), 3.41 min (Method B).

(R)-1-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)-3-hydroxypropan-2-yl (2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)carbamate.

Boc-Linker-Payload: LC/MS (uplc): M+ 982.3, 1.51 min (Method A). 3.8 mg, 0.0041 mmol, 41 %.¹H-NMR (DMSO, 600 MHz): δ 9.09-8.89 (1H, m), 8.55-8.35 (1H, m), 7.84-7.69 (2H, m), 7.45-7.36 (2H, m), 7.36-7.23 (4H, m), 7.16-7.07 (2H, m), 7.05- 6.98 (2H, m), 6.73-6.58 (1H, m), 5.40-5.10 (4H, m), 4.87-4.64 (2H, m), 3.88-3.79 (1H, m), 3.72-3.64 (1H, m), 3.63-3.56 (1H, m), 3.25-3.03 (6H, m), 2.43-2.26 (1H, m), 1.96-1.80 (1H, m), 1.44-1.32 (1H, m), 1.29-1.09 (2H, m), 0.75-0.57 (1H, m). 9.09-8.89 (1H, m), 8.55- 8.35 (1H, m), 7.84-7.69 (2H, m), 7.45-7.36 (2H, m), 7.36-7.23 (4H,

N-((S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-4-((2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxamide.

Boc-linker-payload: LC/MS (uplc): M+ 904.3, 1.29 min (Method A).

31 mg, 0.032 mmol, 85%.¹H-NMR (DMSO, 600 MHz): δ 9.14-8.91 (1H, m), 8.76-8.59 (1H, m), 7.94-7.83 (1H, m), 7.83-7.68 (2H, m), 7.48-7.37 (2H, m), 7.37-7.23 (4H, m), 7.18-7.07 (1H, m), 7.06-6.98 (2H, m), 6.54-6.24 (1H, m), 5.43-5.14 (4H, m), 3.91-3.76 (2H, m), 3.76- 3.65 (1H, m), 3.65-3.54 (1H, m), 3.52-3.28 (4H, m), 3.28-3.13 (3H, m), 3.13-3.01 (1H, m), 2.67-2.47 (2H, m), 2.47-2.31 (1H, m), 2.14-2.00 (1H, m), 2.00-1.86 (1H, m), 1.77-1.66 (2H, m), 1.66-1.57 (2H, m), 1.57-1.47 (1H, m), 1.44-1.35 (1H, m), 1.34-1.10 (4H, m), 1.08-0.99 (3H, m), 0.97-0.83 (2H, m), 0.78-0.60 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 804.2, 0.93 min (Method A). 1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)azetidin-3-yl (2-(2-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 894.2, 1.26 min (Method A).

23 mg, 0.024 mmol, 61 %. ¹H-NMR (DMSO, 600 MHz): δ 9.15-8.97 (1 H, m), 8.77-8.60 (1 H, m), 7.88-7.78 (1 H, m), 7.78-7.69 (1 H, m), 7.43-7.37 (2H, m), 7.37-7.29 (3H, m), 7.28-7.21 (2H, m), 7.14-7.08 (1 H, m), 7.05-6.99 (2H, m), 5.56-5.44 (1 H, m), 5.43-5.21 (2H, ), 5.16- 4.96 (2H, m), 4.46-4.35 (1 H, m), 4.12-4.03 (1 H, m), 4.03-3.95 (1 H, m), 3.88-3.80 (1 H, m), 3.46-3.24 (6H, m), 3.26-3.13 (1 H, m), 3.13-.303 (2H, m), 2.76-2.62 (1 H, m), 2.58-2.34 (3H, m), 1.95-1.80 (1 H, m), 1.68-1.53 (1 H, m), 1.15-0.93 (1 H, m), 0.49-0.30 (1 H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 794.2, 0.91 min (Method A).

1-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)carbamoyl)piperidin-3-yl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate.

Obtain as a mixture of diastereoisomers that was separated before coupling of the linker.

Boc-linker-payload, isolated as pure diastero isomer.

Isomer A, LC/MS (uplc): M+ 922.3, 1.33 min (Method A). Isomer B, LC/MS (uplc): M+ 922.3, 1.34 min (Mehotd A).

Isomer A: 10 mg, 0.010 mmol, 47%.¹H-NMR (DMSO, 600 MHz): δ 9.11-8.93 (1H, m), 8.87-8.69 (1H, m), 7.85-7.74 (1H, m), 7.74-7.64 (1H, m), 7.45-7.36 (2H, m), 7.36-7.28 (2H, m), 7.27-7.18 (2H, m), 7.15-7.06 (1H, m), 7.06-6.97 (3H, m), 5.69-5.59 (1H, m), 5.46-5.36, (1H, m), 5.31-5.13 (1H, m), 4.72-4.62 (1H, m), 4.62-4.54 (1H, m), 3.71-3.60 (2H, m), 3.59- 3.46 (6H, m), 3.46-3.31 (4H, m), 3.31-3.16 (2H, m), 3.15-3.00 (4H, m), 2.99-2.90 (1H, m), 2.89-2.75 (1H, m), 2.48-2.30 (2H, m), 2.29-2.10 (1H, m), 1.94-1.80 (1H, m), 1.79-1.59 (2H, m), 1.58-1.48 (1H, m), 1.47-1.36 (1H, m), 1.19-1.04 (1H, m), 0.89-0.76 (1H, m), 0.55-0.38 (1H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 822.3, 0.96 min (Method A).

Isomer B: 22.5 mg, 0.022 mmol, 52%.¹H-NMR (DMSO, 600 MHz): δ 9.19-8.99 (1H m), 8.92-8.76 (1H, m), 7.81-7.73 (1H, m), 7.71-7.63 (1H, m), 7.45-7.37 (2H, m), 7.37-7.27 (2H, m), 7.26-7.20 (2H, m), 7.14-7.06 (2H, m), 7.05-6.99 (2H, m), 5.58-5.47 (1H, m), 5.42-5.31 (1H, m), 5.25-5.07 (1H, m), 4.69-4.61 (1H, m), 4.56-4.47 (1H, m), 3.90-3.70 (2H, m), 3.65- 3.46 (6H, m), 3.45-3.33 (5H, m), 3.33-3.20 (1H, m), 3.17-2.98 (4H, m), 2.98-2.86 (2H, m), 2.50-2.21 (3H, m), 1.99-1.87 (1H, m), 1.79-1.71 (1H, m), 1.71-1.63 (1H, m), 1.63-1.43 (2H, m), 1.22-1.06 (1H, m), 0.90-0.76 (1H, m), 0.55-0.39 (1H, m). Missing signals hidden under solvent peak. LC/MS (uplc): MH+ 822.3, 0.97 min (Method A). (S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (3-((2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethyl)amino)-3-oxopropyl)carbamate.

Boc-linker-payload: LC/MS (uplc): MH+ 923.2, 1.22 min (Method A).

6.1 mg, 0.006 mmol, 37%. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, m), 8.64 (1 H, m), 8.01 (1 H, m), 7.78 (2H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.12 (1 H, m), 7.01 (2H, m), 6.97 (1 H, m), 6.31 (1 H, m), 5.34 (2H, m), 5.25 (1 H, m), 5.20 (1 H, m), 3.94 (3H, m), 3.80 (1 H, m), 3.75 (1 H, m), 3.50 (1 H, m), 3.45 (2H, m), 3.25 (2H, m), 3.15 (4H, m), 2.59 (1 H, m), 2.50 (1 H, m), 2.26 (1 H, m), 2.17 (2H, m), 2.01 (1 H, m), 1.45 (1 H, m), 1.30-1.20 (2H, m), 1.10 (3H, m), 0.68 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): 823.2, 0.90 min (Method A).

2-amino-3-(((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)-3-oxopropyl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, mixture of diastereosiomers, LC/MS (uplc): M+ 982.3, 1.39 min and 1.40 min (Method A).

Product was obtained as a inseparable mixture of diastereo isomers (4:1 by LC/MS, Method A). 17.8 mg, 0.017 mmol, 70%. ¹H-NMR (DMSO, 600 MHz) Major

diastereoisomer: δ 9.34-9.21 (1 H, m), 9.19-9.05 (1 H, m), 8.71-8.55 (3H, m), 7.94-7.86 (1 H, m), 7.82-7.72 (1 H, m), 7.45-7.37 (2H, m), 7.37-7.31 (2H, m), 7.31-7.26 (1 H, m), 7.26- 7.21 (2H, m), 7.16-7.08 (1 H, m), 7.06-7.01 (2H, m), 5.56-5.49 (1 H, m), 5.41-5.35 (1 H, m), 5.35-5.22 (1 H, m), 5.19-5.07 (1 H, m), 4.56-4.47 (1 H, m), 4.38-4.29 (1 H, m), 4.19-4.10 (1 H, m), 4.00-3.90 (1 H, m), 3.78-3.68 (1 H, m), 3.63-3.54 (3H, m), 3.54-3.48 (3H, m), 3.31-3.22 (1 H, m), 3.22-3.02 (3H, m), 2.83-2.71 (1 H, m), 2.68-2.56 (1 H, m), 2.46-2.35 (1 H, m), 2.02- 1.83 (1 H, m), 1.45-1.33 (1 H, m), 1.28-1.18 (1 H, m), 1.01-0.86 (1 H, m), 0.52-0.36 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 782.3, 0.78 min (Major) and 0.79 min (minor) (Method A).

N-((S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)-3-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1 -yl)propanamide

Boc-linker-payload: LC/MS (uplc): MH+ 836.2, 1.26 min (Method A).

4 mg, 0.004 mmol, 24%. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, m), 8.67 (1 H, m), 8.14 (1 H, m), 7.79 (1 H, m), 7.75 (1 H, m), 7.40 (2H, m), 7.33 (2H, m), 7.27 (2H, m), 7.10 (1 H, m), 7.02 (2H, m), 6.35 (1 H, m), 5.35-5.25 (4H, m), 3.81 (2H, m), 3.75 (1 H, m), 3.62 (3H, m), 3.45 (2H, m), 3.37 (2H, m), 3.16 (2H, m), 3.07 (1 H, m), 2.57 (1 H, m), 2.41 (1 H, m), 2.39 (2H, m), 1.90 (1 H, m), 1.45 (1 H, m), 1.25 (1 H, m), 1.15 (1 H, m), 1.02 (3H, m), 0.70 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 736.2, 0.89 min (Method A).

4-(((S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl)amino)-1-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1-yl)-4-oxobutane-2-sulfonic acid.

Boc-linker-payload, mixture of diastereo isomers: LC/MS (uplc): MH+ 930.5, 1.01 min (Isomer A) and 1.02 min (Isomer B), (Method A). The mixture of diastereoisomers was separated after Boc-deprotection using reverse phase chromatography (PrepLC Method A).

Isomer A, 31 mg, 0.031 mmol, 10%. ¹H-NMR (DMSO, 600 MHz): δ 9.08-8.48 (1 H, m), 8.90-8.75 (1 H, m), 7.94-7.84 (1 H, m), 7.81-7.68 (2H, m), 7.47-7.36 (2H, m), 7.35-7.23 (4H, m), 7.15-7.04 (1 H, m), 7.03-6.94 (2H, m), 6.20-6.08 (1 H, m), 5.46-5.20 (4H, m), 3.95-3.67 (6H, m), 3.29-3.1 1 (2H, m), 2.90-2.64 (4H, m), 2.59-2.44 (3H, m), 2.13-1.91 (1 H, m), 1.78- 1.59 (1 H, m), 1.52-1.36 (1 H, m), 1.35-1.23 (1 H, m), 1.19-1.07 (1 H, m), 1.06-0.94 (3H, m), 0.70-0.53 (1 H, m). Missing signals hidden under the solvent peak. 9.08-8.48 (1 H, m), 8.90-8.75 (1 H, m), 7.94-7.

Isomer B, 35 mg, 0.017 mmol, 1 1 %. ¹H-NMR (DMSO, 600 MHz): δ -NMR (DMSO, 600 MHz): mmol, 1 1 %. m), 7.94-7.84 (1 H, m), 7.81-7.68 (2H, m), 7.47-7.36 (2H, m), 7.35- 7.23 (4H, m), 7.15-7.04 (1 H, m), 7.03-6.94 (2H, m), 6.20-6.08 (1 H, m), 5.46-5.20 (4H, m), 3.95-3.67 (6H, m), 3.29-3.66 (5H, m), 3.29-3.16 (2H, m), 2.84-2.74 (1 H, m), 2.73-2.58 (1 H, m), 2.57-2.42 (5H, m), 2.08-1.97 (1 H, m), 1.71-1.55 (1 H, m), 1.42-1.24 (2H, m), 1.21-1.08 (1 H, m), 1.07-0.95 (3H, m), 0.69-0.51 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 830.5, 0.89 min. (Method A).

(S)-2-(3-((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(1- hydroxycyclopropyl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, LC/MS (uplc): M+ 982.5, 1.64 (Method A). 17.7 mg, 0.018 mmol, 66%. ¹H-NMR (DMSO, 600 MHz): δ 9.20-9.08 (1 H, m), 8.91-8.71 (1 H, m), 7.89-7.81 (1 H, m), 7.81-7.74 (1 H, m), 7.40-7.36 (2H, m), 7.35-7.29 (2H, m), 7.27- 7.23 (2H, m), 7.15-7.09 (1 H, m), 7.05-7.00 (3H, m), 6.38-6.28 (1 H, m), 5.37-5.14 (4H, m), 4.03-3.78 (4H, m), 3.77-3.68 (1 H, m), 3.61-3.52 (2H, m), 3.52-3.45 (2H, m), 3.44-3.29 (4H, m), 3.13-3.03 (2H, m), 3.03-2.93 (1 H, m), 2.57-2.42 (3H, m), 1.16-1.06 (3H, m), 0.69-0.50 (3H, m), 0.50-0.36 (1 H. m). LC/MS (uplc): MH+ 768.3, 0.94 min (Method A).

(S)-2-(3-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-hydroxy-2- methylpropyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, LC/MS (uplc): M+ 984.5, 1.65 min (Method A).

4.1 mg, 0.004 mmol, 54%. ¹H-NMR (DMSO, 600 MHz): δ 9.26-9.05 (1 H, m), 9.00-8.76 (1 H, m), 7.99-7.84 (1 H, m), 7.83-7.71 (1 H, m), 7.51-7.24 (6H, m), 7.19-7.09 (1 H, m), 7.08- 6.95 (3H, m), 6.55-6.37 (1 H, m), 5.50-5.15 (4H, m), 4.09-3.88 (2H, m), 3.88-3.74 (2H, m), 3.60-3.53 (3H, m), 3.53-.344 (3H, m), 3.44-3.23 (4H, ), 3.15-3.01 (2H, m), 2.70-2.59 (1 H, m), 2.39-2.12 (1 H, m), 2.08-1.93 (1 H, m), 1.17-1.09 (3H, m), 1.09-0.98 (3H, m), 0.97-0.78 (3H, m). LC/MS (uplc): MH+ 770.6, 0.95 min (Method A).

(S)-2-(3-((S)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-hydroxytetrahydro-2H- pyran-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, LC/MS (uplc): M+ 1026.6, 1.58 min (Method A).

1 1.3 mg, 0.012 mmol, 32%. ¹H-NMR (DMSO, 600 MHz): δ 9.17-8.95 (1 H, m), 8.77-8.59 (1 H, m), 8.02-7.88 (1 H, m), 7.87-7.74 (1 H, m), 7.45-7.38 (2H, m), 7.38-7.32 (2H, m), 7.32- 7.27 (2H, m), 7.18-7.1 1 (1 H, m), 7.06-6.99 (3H, m), 6.51-6.34 (1 H, m), 5.55-5.10 (4H, m), 4.02-3.88 (4H, m), 3.88-3.71 (2H, m), 3.69-3.60 (1 H, m), 3.60-3.54 (3H, m), 3.54-3.45 (3H, m), 3.44-3.37 (2H, m), 3.36-3.24 (3H, m), 3.16-3.02 (2H, m), 2.72-2.58 (1 H, m), 2.40-2.20 (1 H, m), 2.14-1.93 (1 H, m), 1.52-1.33 (2H, m), 1.21-1.02 (5H, m). LC/MS (uplc): MH+ 812.5, 0.91 min (Method A).

(S)-2-(3-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, LC/MS (uplc): MH+ 884.6, 1.36 min (Method A).25 mg, 0.026 mmol, 82%. ¹H-NMR (DMSO, 600 MHz): δ 9.10-8.93 (1 H, m), 8.78-8.61 (1 H, m), 7.96-7.83 (1 H, m), 7.82-7.70 (1 H, m), 7.48-7.26 (6H, m), 7.20-7.08 (1 H, m), 7.08-6.98 (3H, m), 6.55-6.38 (1 H, m), 5.65-5.45 (1 H, m), 5.45-5.25 (2H, m), 5.25-5.08 (1 H, m), 3.62-3.53 (2H, m), 3.53- 3.45 (2H, m), 3.45-3.35 (2H, m), 3.34-3.17 (2H, m), 3.16-3.05 (2H, m), 3.05-2.92 (3H, m), 2.58-2.39 (2H, m), 2.26-2.02 (1 H, m), 2.02-1.82 (1 H, m), 1.42-1.23 (3H, m), 1.23-1.06 (3H, m), 0.92-0.73 (3H, m). LC/MS (uplc): MH+ 784.4, 1.01 min (Method A).

(3S,4S)-4-(((S)-N-((S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-methoxy-2- methylpropyl)-2-hydroxypropanamido)methyl)pyrrolidin-3-yl (2-(2-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate.

Boc-linker-payload, LC/MS (uplc): MH+895.3, 1.37 min (Method A).

26.8 mg, 0.031 mmol, 79%. ¹H-NMR (DMSO, 600 MHz): δ 9.06-8.90 (1 H, m), 8.88-8.73 (1 H, m), 8.72-8.63 (1 H, m), 7.96-7.91 (1 H, m), 7.90-7.69 (2H, m), 7.45-7.30 (8H, m), 7.29- 7.24 (1 H, m), 7.19-7.07 (3H, m), 7.06-6.98 (3H, m), 6.00-5.84 (1 H, m), 5.62-5.32 (2H, m), 5.17-5.03 (2h, m), 4.92-4.82 (2H, m), 4.81-4.70 (3H, m), 4.62-4.49 (2H, m), 4.1 1-3.99 (1 H, m), 3.97-3.86 (2H, m), 3.83-3.73 (1 H, m), 3.62-3.45 (7H, m), 3.44-3.28 (4H, m), 3.22-3.05 (5H, m), 3.04-2.96 (1 H, m), 2.92-2.71 (4H, m), 2.43-2.33 (1 H, m),2.04-1.85 (2H, m), 1.66- 1.51 (m, 1 H), 1.50-1.41 (1 H, m), 1.36-1.30 (3H, m), 1.30-1.24 (3H, m), 1.08-0.98 (1 H, m), 0.89-0.79 (2H, m), 0.77-0.65 (3H, m). Mixture of rotamers can be observed by NMR.

LC/MS (uplc): MH+ 753.3, 0.98 min (Method A). (S)-2-((((R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutoxy)carbonyl)amino)-3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoic acid

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 3 days) after purification by reverse phase column chromatography (8 mg, 17%, as a TFA salt).

LC/MS (Method B): [M+H]⁺ 769.4, Rt 3.21 min. ¹H-NMR (DMSO, 400 MHz): δ 12.89 (1 H, br s), 8.97 (1 H, br s), 8.54 (1 H, br s), 7.97 (1 H, d, 3.9 Hz), 7.80-7.70 (1 H, m), 7.45-7.30 (7H, m), 7.17-7.08 (1 H, m), 6.99 (2H, s), 5.84 (1 H, s), 5.40 (1 H, d, 15.3 Hz), 5.25 (1 H, d, 56 Hz), 5.06 (1 H, d, 15.3 Hz), 4.63-4.54 (1 H, m), 4.21 - 4.1 1 (1 H, m), 4.06-3.88 (2H, m), 3.80-3.63 (4H, m), 3.34-3.23 (2H, m), 2.40-2.35 (1 H, m), 2.00-1.85 (1 H, m), 1.58-1.46 (1 H, m), 1.35 (3H, d, 6.2 Hz), 1.32-1.21 (1 H, m), 0.95 (3H, s), 0.71 (3H, s). 1 H is hidden under DMSO, OH not seen.

Under N2: To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (40 mg, 0.057 mmol) in DMF 1 mL was added DIPEA (0.299 mL, 1.712 mmol) followed by bis(4-nitrophenyl) carbonate (39.9 mg, 0.131 mmol). Reaction mixture was stirred at room temperature for 16 h. Upon completion by LC/MS (uplc), was added 1-(2-((2-Hydroxyethyl)amino)ethyl)-1 H-pyrrole-2,5-dione (Linker 1 , TFA salt) (66.1 mg, 0.1 14 mmol), and the reaction mixture was stirred at room temperature for 16 h. Another 1 -(2-((2-Hydroxyethyl)amino)ethyl)-1 H-pyrrole-2,5-dione (Linker 1 , TFA salt) (66.1 mg, 0.1 14 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. Ethyl acetate and cold water were added, organic layer was washed twice with brine, dried with Na2SC>4, filtered and solvent was removed in vacuo. Absorbed onto Isolute. The desired product was obtained after purification by column chromatography (4 g silica gel, 0 to 100 % EtOAc in Heptane, 18 mg, 0.017 mmol, 29%, purity 85%) as a colorless oil.

LC/MS (Method A): [M+H]⁺ 91 1 .5, Rt 1 .27 min.

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3-dimethylbutyl (2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)(2-hydroxyethyl)carbamate

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-4-(((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)(2-hydroxyethyl)carbamoyl)oxy)- 2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (18 mg, 0.017 mmol) was dissolved in a mixture of acetonitrile (1 ml_) and water (0.500 ml_). TFA (0.065 ml_, 0.840 mmol) was added and reaction mixture was stirred at 60^°C for seven days. The desired product was obtained after purification by reverse phase column chromatography (9 mg, 59%, as a TFA salt).

LC/MS (Method B): [M+H]⁺ 769.5, Rt 3.35 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers ratio 1 :1 ): δ 9.02 (1 H, br s), 8.68 (1 H, br s), 7.98 (1 H, s), 7.82-7.73 (1 H, m), 7.44-7.31 (6H, m), 7.16-7.10 (1 H, m), 6.97 and 6.96 (2H, two singlets, rotamers), 5.84 and 5.84 (1 H, two singlets, rotamers), 5.41 (1 H, d, 15.4 Hz), 5.27 (1 H, d, 54 Hz), 5.06 (1 H, d, 15.4 Hz), 4.62-4.57 (1 H, m), 4.05-3.90 (2H, m), 3.82-3.67 (2H, m), 3.57-3.49 (2H, m), 3.46-3.07 (8H, m), 2.42-2.36 (1 H, m), 1.98-1.85 (2H, m), 1.58-1.49 (1 H, m), 1.38-1.23 (4H, m), 1.01 and 0.97 (3H, two singlets, rotamers), 0.69 and 0.67 (3H, two singlets, rotamers).

(3R,4R)-tert-butyl 3-((R)-3-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-14-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-2-((S)-2-hydroxypropanoyl)-4,4-dimethyl-8-oxo- 7,12-dioxa-2,9-diazatetradecyl)-4-fluoropyrrolidine-1-carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. Not purified. Colorless oil (40 mg, 0.023 mmol, 55% yield, 52% pure).

LC/MS (Method A): [M+H]⁺ 912.2, Rt 1.34 min.

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3-dimethylbutyl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 29 h). Purified by reverse phase column chromatography, colorless solid (19 mg, 0.022 mmol, 94% yield, TFA salt). LC/MS (Method B): [M+H]⁺ 769.3, Rt 3.79 min. ¹H-NMR (DMSO, 600 MHz): δ 9.04 (1 H, br s), 8.73 (1 H, br s), 7.97-7.93 (1 H, m), 7.78-7.72 (1 H, m), 7.44-7.30 (6H, m), 7.16-7.09 (1 H, m), 7.00 (2H, s), 6.93-6.88 (1 H, m), 5.84 (1 H, s), 5.40 (1 H, d, 15 Hz), 5.25 (1 H, d, 54 Hz), 5.06 (1 H, d, 15 Hz), 4.62-4.56 (1 H, m), 4.05-3.65 (4H, m), 3.45-3.40 (2H, m), 3.37-3.15 (2H, m), 3.12-3.05 (2H, m), 2.42-2.33 (1 H, m), 2.00-1.85 (2H, m), 1.60-1.50 (1 H, m), 1.35 (3H, d, 5.9 Hz), 1.32-1.24 (1 H, m), 0.93 (3H, s), 0.73 (3H, s).

(3R,4R)-tert-butyl 3-((R)-2-((S)-2-acetoxypropanoyl)-3-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-32-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,4- dimethyl-8-oxo-7,12,15,18,21, 24,27, 30-octaoxa-2,9-diazadotriacontyl)-4- fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. Not purified. Colorless oil (28 mg, 0.024 mmol, crude).

LC/MS (Method A): [M+H]⁺ 1 175.4, [M+NH₄]⁺ 1 192.5, Rt 1.34 min. (R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3-dimethylbutyl (23-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3,6,9,12,15,18,21 -heptaoxatricosyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase column chromatography. Colorless solid as a TFA salt (2.3 mg, 2.005 μιηοΙ, 9% yield, 100% pure).

LC/MS (Method B): [M+H]⁺ 1033.3; Rt 4.02 min.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-

4-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

Purified by column chromatography (40 g silica gel, 0 to 100 % EtOAc in Heptane).

Colorless oil (500 mg, 0.604 mmol, 60% yield, 96% pure).

LC/MS (Method A): [M+H]⁺ 795.6, Rt 1.20 min.

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3R,4R)-1-(tert- butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanoic acid (980 mg, 1.005 mmol) and N-(2-aminoethyl)maleimide (TFA salt, 51 1 mg, 2.010 mmol) were dissolved in DMF (30 ml) and DIPEA (0.878 ml, 5.03 mmol), followed by HATU (573 mg, 1.508 mmol) were added. Reaction mixture stirred at RT for 1 h. Reaction mixture was diluted with EA and washed with brine (three times). The combined organic layers were dried with Na₂S0₄, filtered and concentrated. Residue was purified by column chromatography (40 g, silica gel, 0 to 100 % EtOAc in Heptane) to provide colorless oil (500 mg, 0.604 mmol, 60% yield, 96% pure).

LC/MS (Method A): [M+H]⁺ 795.6, Rt 1.20 min.

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-N-(2-(2,5-dioxo-2,5-dihydro-

1 H-pyrrol-1-yl)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamido)-3,3-dimethylbutanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography, colorless solid (1 12 mg, 0.137 mmol, 55 % yield as a TFA salt).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4- ((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (200 mg, 0.252 mmol) was dissolved in DCM (4 ml). TFA (1.94 ml, 25.2 mmol) was added and the reaction mixture was stirred at RT for 1 h. Concentrated and purified by reverse phase chromatography to yield colorless solid (1 12 mg, 0.137 mmol, 55 % yield as a TFA salt).

LC/MS (Method B): [M+H]⁺ 695.4, Rt 3.02 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers ratio 5:1 , some peaks for the minor rotamer can't be unambiguously assigned): δ 8.96 (1.2H, br s), 8.66 (1.2H, br s), 7.85-7.80 (2H, m), 7.80-7.75 (1 H, m), 7.44-7.31 (6.8H, m), 7.16-7.09 (1.6H, m), 7.01 (0.4H, s), 6.99 (2H, s), 6.17 (1 H, s), 5.72 (0.2H, s), 5.55 (0.2H, d, 15.9 Hz), 5.46 (0.2H, d, 15.9 Hz), 5.33 (1 H, d, 15.0 Hz), 5.17-5.05 (2.2H, m), 4.80-4.74 (0.2H, m), 4.57-4.50 (1 H, m), 4.47-4.41 (0.2H, m), 4.04-3.99 (0.4H, m), 3.95-3.90 (2H, m), 3.41 (2H, t, 6.0 Hz), 3.34-2.98 (5.2H, m), 2.31-2.22 (1.2H, m), 2.12 (1 H, d, 13.8 Hz), 2.08-2.02 (0.2H, m), 1.99-1.89 (1.2H, m), 1.82 (1 H, d, 13.8 Hz), 1.73- 1.58 (1 H, m), 1.35 (3H, d, 6.0 Hz), 1.1 1 (0.6H, s), 1.04 (0.6H, s), 1.00 (3H, s), 0.92 (3H, s), 0.77 (0.6H, d, 6.0 Hz).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 4-((2-(2-(2,5-dioxo-2,5-dihydro-1 H^yrrol-1-yl)ethoxy)ethyl)amino)-2,2-dimethyl-4- oxobutyl)-2-hydroxypropanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

Purified by column chromatography (24 g silica gel, 0 to 100 % EtOAc in Heptane). Colorless oil (108 mg, 0.109 mmol, 44% yield, 85% pure).

LC/MS (Method A): [M+H]⁺ 839.5, Rt 1.21 min.

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-N-(2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)-2-hydroxypropanamido)-3,3-dimethylbutanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography, colorless solid as a TFA salt (23 mg, 0.026 mmol, 48 % yield). LC/MS (Method B): [M+H]⁺ 739.4, Rt 3.20 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers ratio 5: 1 , some peaks for the minor rotamer can't be clearly identified): δ 8.93 (1 .2H, br s), 8.61 (1 .2H, br s), 7.86-7.76 (2.6H, m), 7.72-7.67 (1 H, m), 7.60-7.56 (0.2H, m), 7.46-7.30 (7.2H, m), 7.16-7.09 (1 .8H, m), 6.99 (2.4H, br s), 6.19 (1 H, s), 5.69 (0.2H, s), 5.58 (0.2H, d, 15.0 Hz), 5.48 (0.2H, d, 15.0 Hz), 5.33 (1 H, d, 15.0 Hz), 5.20-5.05 (2.2H, m), 4.81 -4.75 (0.2H, m), 4.58-4.51 (1 .2H, m), 4.05-3.90 (2.4H, m), 3.37-3.25 (7.2H, m), 3.15-3.00 (5.2H, m), 2.60-2.55 (1 .2H, m), 2.32-2.25 (2H, m), 2.21 (1 H, d, 13.8 Hz), 2.12-2.02 (0.4H, m), 2.00-1 .90 (2.2H, m), 1 .74-1 .60 (1 H, m), 1 .35 (3H, d, 6.6 Hz), 1 .13 (0.6H, s), 1 .07 (0.6H, s), 1 .04 (3H, s), 0.93 (3H, s), 0.77 (0.6H, d, 5.4 Hz).

3-((2-(2,5-dioxo-2,5-dihydro-1 H^yrrol-1-yl)ethyl)amino)-2,2-dimethyl-3-oxopropyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

No purification. Crude compound

LC/MS (Method A): [M+H]⁺ 781 .4, Rt 1 .19 min.

(R)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-N-(2-(2,5-dioxo-2,

1 H-pyrrol-1-yl)ethyl)-3-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamido)-2,2-dimethylpropanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography, colorless solid as a TFA salt (2.4 mg, 3.02 μιηοΙ, 7% yield, 100% pure). LC/MS (Method B): [M+H]⁺ 681.2; Rt 3.13 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks for the major rotamer are reported): δ 8.95 (1 H, br s), 8.67 (1 H, br s), 7.84-7.73 (2H, m), 7.65 (1 H, d, 3.7Hz), 7.45-7.28 (6H, m), 7.15-7.05 (1 H, m), 6.94 (2H, s), 6.42 (1 H, s), 5.22-5.18 (2H, m), 5.16-5.07 (1 H, m), 4.57-4.51 (1 H, m), 3.95-3.85 (2H, m), 3.40-3.25 (3H, m), 3.15-3.00 (3H, m), 2.30-2.22 (1 H, m), 1.97-1.87 (1 H, m), 1.80-1.65 (1 H, m), 1.34-1.30 (6H, m), 1.02 (3H, s).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 3-((2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)amino)-2,2-dimethyl-3- oxopropyl)-2-hydroxypropanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

No purification. Crude compound LC/MS (Method A): [M+H]⁺ 825.4, Rt 1.20

(R)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-N-(2-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)-3-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)-2-hydroxypropanamido)-2,2-dimethylpropanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography, colorless solid as a TFA salt (2.4 mg, 2.86 μιηοΙ, 6% yield, 100% pure).

LC/MS (Method B): [M+H]⁺ 725.3; Rt 3.28 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks for the major rotamer are reported): δ 8.93 (1 H, br s), 8.63 (1 H, br s), 7.83-7.72 (1 H, m), 7.71-7.66 (2H, m), 7.46-7.26 (6H, m), 7.13-7.06 (1 H, m), 6.99 (2H, s), 6.43 (1 H, s), 5.25-5.17 (2H, m), 5.16-5.03 (1 H, m), 4.57-4.51 (1 H, m), 3.95-3.83 (2H, m), 3.46-3.39 (2H, m), 3.34-3.21 (3H, m), 3.19-3.01 (4H, m), 2.98-2.86 (1 H, m), 2.30-2.22 (1 H, m), 1.97-1.87 (1 H, m), 1.75-1.60 (1 H, m), 1.40 (3H, s), 1.34 (3H, d, 6.2Hz), 1.02 (3H, s). (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-3-(((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)carbamoyl)oxy)-2,2- dimethylpropyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. Not purified. Colorless oil (27 mg, 0.032 mmol, crude).

LC/MS (Method A): [M+H]⁺ 853.4, Rt 1.33 min.

(R)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-2,2-dimethylpropyl (2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 17 h). Colorless solid as a TFA salt (4 mg, 4.85 pmol, 15% yield, 100% pure).

LC/MS (Method B): [M+H]⁺ 71 1.3; Rt 3.59 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, peaks for the major rotamer are reported): δ 9.01 (1 H, br s), 8.73 (1 H, br s), 7.90 (1 H, d, 3.8Hz), 7.80-7.74 (1 H, m), 7.44-7.30 (6H, m), 7.16-7.08 (2H, m), 6.99 (2H, s), 6.04 (1 H, s), 5.36 (1 H, d, 15.2Hz), 5.22-5.04 (2H, m), 4.59-4.54 (1 H, m), 4.05-3.85 (2H, m, overlaps with water peak), 3.80-3.72 (2H, m), 3.45 (2H, t, 6.0 Hz), 3.40-3.25 (2H, m), 3.14-3.08 (2H, m), 2.36-2.28 (1 H, m), 2.00-1.90 (1 H, m), 1.80-1.68 (1 H, m), 1.34 (3H, d, 6.2Hz), 0.87 (3H, s), 0.80 (3H, s).

(3R,4R)-tert-butyl 3-((R)-2-((S)-2-acetoxypropanoyl)-3-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,4- dimethyl-7-oxo-6,11-dioxa-2,8-diazatridecyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. Not purified. Colorless oil (37 mg, 0.042 mmol, crude).

LC/MS (Method A): [M+H]⁺ 897.5, Rt 1.34 min.

(R)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-3-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-2,2-dimethylpropyl (2-(2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 16 h). Purified by reverse phase chromatography to yield desired product. Colorless solid as a TFA salt (7.7mg, 7.98 μιτιοΙ, 19% yield, 90% pure).

LC/MS (Method B): [M+H]⁺ 755.3; Rt 3.60 min. ¹H-NMR (DMSO, 600 MHz mixture of rotamers, peaks for the major rotamer are reported): δ 9.00 (1 H, br s), 8.70 (1 H, br s), 7.90 (1 H, d, 3.9Hz), 7.84-7.74 (1 H, m), 7.45-7.32 (6H, m), 7.16-7.09 (1 H, m), 7.01 (2H, s), 6.94 (1 H, t, 5.9Hz), 6.07 (1 H, s), 5.37 (1 H, d, 15.3Hz), 5.21-5.06 (2H, m), 4.60-4.53 (1 H, m), 4.07-3.85 (2H, m), 3.80-3.70 (2H, m, overlaps with water peak), 3.60-3.22 (7H, m, overlaps with water peak), 3.20-3.00 (3H, m), 2.35-2.27 (1 H, m), 2.00-1.90 (1 H, m), 1.85-1.70 (1 H, m), 1.34 (3H, d, 6.1 Hz), 0.91 (3H, s), 0.83 (3H, s).

Step 1 : (3R,4R)-tert-butyl 3-((R)-18-azido-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H- imidazol-2-yl)-2-((S)-2-hydroxypropanoyl)-4,4-dimethyl-6-oxo-10,13,16-trioxa-2,7- diazaoctadecyl)-4-fluoropyrrolidine-1 -carboxylate

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3R,4R)-1-(tert- butoxycarbonyl)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanoic acid (60 mg, 0.043 mmol) and 1 1-azido-3,6,9trioxaundecan-1-amine (0.017 ml, 0.086 mmol) were dissolved in DMF (2 ml) and DIPEA (0.037 ml, 0.214 mmol), followed by HATU (24.42 mg, 0.064 mmol) were added. Reaction mixture stirred at room temperature for 1 h.

Diluted with EA and washed with brine (^*3). The combined organic layers were dried and concentrated to give 37 mg (100% yield) of desired product, which was used in the next step without further purification. LC/MS (Method A): [M+H]⁺ 873.5, Rt 1.27 min.

Step 2: (3R.4R)-tert-butvl 3-((R)-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 18-(4-((2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)-1 H-1 ,2,3-triazol-1 -yl)-2-((S)-2- hydroxypropanoyl)-4,4-dimethyl-6-oxo-10,13,16-trioxa-2,7-diazaoctadecyl)-4- fluoropyrrolidine-1 -carboxylate

To (3R,4R)-tert-butyl 3-((R)-18-azido-3-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)- 2-((S)-2-hydroxypropanoyl)-4,4-dimethyl-6-oxo-10,13,16-trioxa-2,7-diazaoctadecyl)-4- fl uo ro pyrrol id ine-1 -carboxylate (step 1 ) (37 mg, 0.043 mmol) and 1-(prop-2-yn-1-yl)-1 H- pyrrole-2,5-dione (9.94 mg, 0.074 mmol) in Acetonitrile (1 mL) a solution of Cul (9.34 mg, 0.049 mmol) in Water (1 mL) was added followed by TEA (6.83 μΙ, 0.049 mmol). Reaction mixture was stirred at room temperature for 60 h. The reaction mixture was filtered, diluted with ethyl acetate, washed with brine and dried over Na₂S0₄. Purified by column chromatography (12 g silica gel, 0 to 20 % DCM in Methanol). Colorless oil (24 mg, 0.013 mmol, 27% yield, 55% pure).

LC/MS (Method A): [M+H]⁺ 1008.5, Rt 1.15 min.

Step 3: (R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-N-(2-(2-(2-(2-(4-((2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)methyl)-1 H-1, 2,3-triazol-1- yl)ethoxy)ethoxy)ethoxy)ethyl)-4-((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hydroxypropanamido)-3,3-dimethylbutanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography to yield desired product (2.8 mg, 2.60 μιηοΙ, 21.68 % yield, TFA salt) as colorless solid.

LC/MS (Method B): [M+H]⁺ 908.5, Rt 3.08 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers ratio 5:1 , some peaks for the minor rotamer can't be unambiguously assigned): δ 8.97 (1.2H, br s), 8.69 (1.2H, br s), 7.96 (1.2H, s), 8.03-7.93 (0.2H, m), 7.85-7.75 (3.4H, m), 7.59-7.55 (0.2H, m), 7.45-7.29 (7.4H, m), 7.16-7.09 (1.6H, m), 7.07 (2H, s), 6.21 (1 H, s), 5.72 (0.2H, s), 5.59 (0.2H, d, 16.2 Hz), 5.48 (0.2H, d, 16.2 Hz), 5.34 (1 H, d, 15.0 Hz), 5.20-5.06 (2.2H, m), 4.82-4.76 (0.2H, m), 4.65 (2.4H, s), 4.57-4.52 (1 H, m), 4.50-4.44 (2.8H, m), 4.07-4.02 (0.2H, m), 3.97-3.93 (2H, m), 3.38 (2.4H, t, 5.1 Hz), 3.52-3.40 (10H, m), 3.32-3.25 (2.4H, m), 3.17-3.12 (2.4H, m), 3.07-3.02 (0.4H, m), 2.32-2.25 (1.2H, m), 2.22 (1 H, d, 15.0 Hz), 2.12-2.01 (0.6H, m), 2.00-1.90 (2.2H, m), 1.75-1.60 (1 H, m), 1.36 (3H, d, 6.0 Hz), 1.15 (0.6H, s), 1.09 (0.6H, s), 1.06 (3H, s), 0.94 (3H, s), 0.78 (0.6H, d, 6.0 Hz).

(3R,4R)-tert-butyl 3-((S)-2-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methyl)-14-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5- methyl-3,8-dioxo-7,12-dioxa-2,4,9-triazatetradecyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. Purified by flash

chromatography (24 g, silica gel) eluting with DCM/MeOH. Colorless oil (70 mg, 0.077 mmol, 51 % yield).

LC/MS (Method A): [M+H]⁺ 910.4, Rt 1.35 min.

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro- 2H-pyran-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (2-(2- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)ethyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography to yield desired product (35 mg, 0.038 mmol, 49 % yield, TFA salt) as a colorless solid.

LC/MS (Method B): [M+H]⁺ 810.2, Rt 4.10 min. ¹H-NMR (DMSO, 400 MHz): δ 8.94 (1 H, bs), 8.48 (1 H, bs), 7.92 (1 H, d, 3.9Hz), 7.78-7.70 (1 H, m), 7.45-7.25 (7H, m), 7.15-7.07 (1 H, m), 7.03 (2H, s), 6.45 (1 H, d, 7.4Hz), 5.56 (1 H, s), 5.39 (1 H, d, 15.3Hz), 5.30-5.07 (2H, m), 4.05-3.85 (4H, m), 3.84-3.70 (1 H, m), 3.60-3.45 (6H, m), 3.43-3.20 (6H, m), 3.12- 3.02 (2H, m), 2.47-2.37 (1 H, m), 2.30-2.10 (1 H, m), 1.82-1 .67 (1 H, m), 1 .54-1 .35 (2H, m), 1 .30-1 .20 (1 H, m), 1 .17-1 .05 (6H, m), 0.97-0.89 (1 H, m).

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanamido)-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Not purified (36 mg, purity 38%, crude, yield 69%, 0.016 mmol).

LC/MS (Method A): [M+H]⁺ 851 .5; Rt 1 .25 min.

(S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-(3-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)propanamido)-2,2-dimethylbutyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 20 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (6 mg, 6.82 μιηοΙ, 43% yield, 92% pure).

LC/MS (Method B): [M+H]⁺ 709.4; Rt 3.27 min. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, br s), 8.75 (1 H, br s), 7.93 (1 H, d, 3.8Hz), 7.80-7.74 (1 H, m), 7.74-7.66 (1 H, m), 7.44-7.30 (6H, m), 7.16-7.09 (1 H, m), 6.99 (2H, s), 5.84 (1 H, s), 5.38 (1 H, d, 15.4Hz), 5.25-5.19 (1 H, m), 5.09 (1 H, d, 15.4Hz), 4.63-4.55 (1 H, m), 4.05-3.90 (2H, m), 3.58-3.53 (2H, m, overlaps with water peak), 3.35-3.15 (2H, m), 2.86-2.78 (2H, m), 2.41-2.35 (1 H, m), 2.31- 2.23 (2H, m), 1.97-1.85 (2H, m), 1.41 -1.32 (4H, m), 1.17-1.06 (1 H, m), 0.92 (3H, s), 0.79 (3H, s).

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)-4-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)propanamido)-

2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Not purified (33 mg, yield 100%, crude, 0.037 mmol).

LC/MS (Method A): [M+H]⁺ 895.5; Rt 1.26 min.

(S)-N-((R)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-(3-(2-(2,5-dioxo-2,5- dihydro-1 H-pyrrol-1-yl)ethoxy)propanamido)-2,2-dimethylbutyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (60^°C, 20 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (10 mg, 1 1.02 μιηοΙ, 30% yield, 97% pure).

LC/MS (Method B): [M+H]⁺ 753.4; Rt 3.33 min. ¹H-NMR (DMSO, 600 MHz): δ 9.05 (1 H, br s), 8.75 (1 H, br s), 7.93 (1 H, d, 3.6 Hz), 7.80-7.74 (1 H, m), 7.57-7.52 (1 H, m), 7.45- 7.30 (6H, m), 7.16-7.07 (1 H, m), 7.01 (2H, s), 5.84 (1 H, s), 5.38 (1 H, d, 15.3Hz), 5.32- 5.17 (1 H, m), 5.09 (1 H, d, 15.3Hz), 4.63-4.55 (1 H, m), 4.05-3.85 (2H, m, overlaps with water peak), 3.55-3.51 (4H, m), 3.47-3.43 (2H, m), 3.40-3.15 (2H, m), 2.90-2.80 (2H, m), 2.42-2.32 (1 H, m), 2.23-2.16 (2H, m), 1.92-1.88 (1 H, m), 1.45-1.30 (4H, m), 1.20-1.10 (1 H, m), 0.93 (3H, s), 0.80 (3H, s).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)propanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Purified by flash chromatography (24 g, silica gel) eluting with DCM/MeOH. Colorless oil (50 mg, 0.059 mmol, 72% yield).

LC/MS (Method A): [M+H]⁺ 849.2; Rt 1.26 min.

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 6 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (12 mg, 0.013 mmol, 45% yield, 95% pure).

LC/MS (Method B): [M+H]⁺ 749.2; Rt 3.27 min. ¹H-NMR (DMSO, 600 MHz, mixture of rotamers, 120^°C): δ 7.83-7.77 (1 H, m), 7.67 (1 H, d, 3.7 Hz), 7.45-7.32 (3H, m), 7.30-7.15 (3H, m), 7.10-7.07 (1 H, m), 6.93 (2H, s), 5.40-5.10 (4H, m), 4.40-4.15 (1 H, m), 4.05-3.85 (4H, m), 3.76-3.62 (4H, m), 3.55-3.25 (7H, m), 2.95-2.85 (1 H, m), 2.75-2.55 (5H, m), 2.37- 2.20 (1 H, m), 1.55-1.25 (3H, m), 1.05-0.85 (1 H, m).

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H^yran-4-yl)methyl)-4-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)ethoxy)propanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1- carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Purified by flash chromatography (12 g, silica gel) eluting with DCM/MeOH. Yellow oil (30 mg, 0.034 mmol, 57% yield).

LC/MS (Method A): [M+H]⁺ 893.3; Rt 1.27 min.

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-4-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethoxy)propanoyl)-N-

(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 4 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (22 mg, 0.024 mmol, 72% yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 793.1 ; Rt 3.56 min.

(3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-4-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Purified by flash chromatography (4 g, silica gel) eluting with DCM/MeOH. Yellow oil (13 mg, 0.014 mmol, 35% yield).

LC/MS (Method A): [M+H]⁺ 891.3; Rt 1.31 min.

(S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-4-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 6 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (10 mg, 10.72 μιηοΙ, 74% yield, 97% pure).

LC/MS (Method B): [M+H]⁺ 791.3; Rt 3.72 min.

(3R,4R)-tert-butyl 3-(((R)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)propanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Purified by flash chromatography (12 g, silica gel) eluting with DCM/MeOH. Colorless oil (15 mg, 0.018 mmol, 60% yield).

LC/MS (Method A): [M+H]⁺ 849.2; Rt 1.30 min.

(R)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 3 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (15 mg, 0.017 mmol, 95% yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 749.1 ; Rt 3.56 min.

(3R,4R)-tert-butyl 3-(((R)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-4-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)hexanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Amine. Purified by flash chromatography (4 g, silica gel) eluting with DCM/MeOH. Yellow oil (22 mg, 0.025 mmol, 84% yield).

LC/MS (Method A): [M+H]⁺ 891.2; Rt 1.33 min.

(R)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(tetrahydro-2H-pyran-4- yl)methyl)-4-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)morpholine-2-carboxamide

The title compound was prepared by a method similar to that described in General

Method for Boc-Deprotection of Linker-Payload Combinations (RT, 6 h). Purified by reverse phase chromatography to afford colorless solid as a TFA salt (15 mg, 0.017 mmol, 66% yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 791.2; Rt 3.88 min.

(3R,4R)-tert-butyl 3-((S)-2-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)(4- methyltetrahydro-2H-pyran-4-yl)methyl)-32-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-5- methyl-3,8-dioxo-7,12,15,18,21, 24,27, 30-octaoxa-2,4,9-triazadotriacontyl)-4- fluoropyrrolidine-1 -carboxylate

The title compound was prepared by a method similar to that described in Payload Attachment to Linker Component at a Payload Hydroxyl. No purification. Yellow oil (63 mg, 0.054 mmol, 83% yield, crude).

LC/MS (Method A): [M+H]⁺ 1 174.6, ), [M+NH₄]⁺ 1 191.7, Rt 1.35 min.

(S)-2-(3-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)(4-methyltetrahydro- 2H-pyran-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)ureido)propyl (23- (2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-3,6,9,12,15,18,21 -heptaoxatricosyl)carbamate

The title compound was prepared by a method similar to that described in General Method for Boc-Deprotection of Linker-Payload Combinations (RT, 1 h). Purified by reverse phase chromatography to yield desired product (10 mg, 8.33 μιηοΙ, 16 % yield, TFA salt) as a colorless solid.

LC/MS (Method Bv2): [M+H]⁺ 1074.4, Rt 4.49 min.

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (3-(N-((R)-1-(3-benzyl-7-chloro-4- oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-4- methylbenzamido)propyl)carbamate

The title compound was prepared by a method similar to that described in General Procedure for attaching Linkers to Amines in Payload compounds of Formula (II). Purified by reverse phase chromatography to afford colorless solid (8 mg, 6.74 μιηοΙ, 14% yield, 94% pure).

LC/MS (Method B): [M+H]⁺ 1 1 15.6; Rt 5.71 min. ¹H-NMR (DMSO, 400 MHz): δ 9.96 (1 H, s), 8.23 (1 H, d, J = 8.6 Hz), 8.07 (1 H, d, J = 7.5 Hz), 7.83 - 7.74 (2H, m), 7.65 (1 H, dd, J = 8.6, 2.1 Hz), 7.61 - 7.54 (2H, m), 7.41 - 7.10 (12H m), 7.00 (2H, s), 6.77 (1 H, t, J = 5.9 Hz), 5.97 (2H, br s), 5.88 (1 H, d, J = 16.3 Hz), 5.53 (1 H, d, J = 10.5 Hz), 5.07 (1 H, d, J = 16.3 Hz), 4.89 - 4.75 (2H, m), 4.43 - 4.33 (1 H, m), 4.19 (1 H, dd, J = 8.6, 6.7 Hz), 3.40- 3.20 (6H, m), 3.08 - 2.90 (2H, m), 2.79 - 2.68 (1 H, m), 2.46-2.40 (2H, m), 2.31 (3H, s), 2.24-2.04 (2H, m), 2.02-1.90 (1 H, m), 1.76-1.10 (10H, m), 0.92 - 0.79 (9H, m), 0.48 (3H, d, J = 6.4 Hz).

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (3-(N-((R)-1-(3-benzyl-7-chloro-4- oxo-4H-chromen-2-yl)-2-methylpropyl)-4-methylbenzamido)propyl)carbamate

The title compound was prepared by a method similar to that described in General Procedure for attaching Linkers to Amines in Payload compounds of Formula (II). Purified by reverse phase chromatography to afford colorless solid (26mg, 23 μιηοΙ, 38 % yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 1 1 15.5; Rt 5.58 min. ¹H-NMR (DMSO, 400 MHz): δ 9.96 (1 H, s), 8.1 1-8.04 (2H, m), 7.90 (1 H, s), 7.83-7.76 (1 H, d, J = 8.6 Hz), 7.61-7.52 (3H, m), 7.29- 7.1 1 (12H, m), 7.00 (2H, s), 5.99 (2H, br s), 5.76 (1 H, d, J = 10.0 Hz), 4.90-4.77 (2H, m), 4.42-4.32 (1 H, m), 4.23-4.12 (2H, m), 3.95-3.85 (1 H, m), 3.42-3.25 (3H, m), 3.06-2.88 (2H, m), 2.64-2.53 (6H, m), 2.31 (3H, s), 2.25-2.05 (1 H, m), 2.02-1.87 (1 H, m), 1.75-1.65 (1 H, m), 1 .65-1 .29 (7H, m), 1 .25-1 .10 (3H, m), 0.97 (3H, d, J = 6.6 Hz), 0.87-0.79 (6H, m), 0.57-0.50 (3H, m).

Under N₂: To a solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (50 mg, 0.071 mmol) in DMF 5 mL was added DIPEA (0.374 mL, 2.140 mmol) followed by bis(4-nitrophenyl) carbonate (50 mg, 0.164 mmol). Reaction mixture was stirred at room temperature for 16 h. Upon completion by LC/MS (uplc), was added 1-(2-((2-Hydroxyethyl)amino)ethyl)-1 H-pyrrole-2,5-dione (Linker 1 , TFA salt) (180 mg, 0.604 mmol), and the reaction mixture was stirred at room temperature for 16 h. Ethyl acetate and cold water were added, organic layer was washed twice with brine, dried with Na₂S0₄, filtered and solvent was removed in vacuo. Absorbed onto Isolute. The desired product was obtained after purification by column chromatography (4 g silica gel, 0 to 100 % EtOAc in Heptane, 9 mg, 0.071 mmol, 14%) as a colorless oil. LC/MS (Method A): MH+ 91 1 .7, 1 .26 min..

(3R,4R)-tert-butyl 3-((R)-2-((S)-2-acetoxypropanoyl)-3-(1-benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-9-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)- 4,4,14,14-tetramethyl-8,12-dioxo-7,13-dioxa-2,9-diazapentadecyl)-4- fluoropyrrolidine-1 -carboxylate

Under N₂: To a solution of (3R,4R)-tert-but l 3-(((S)-2-acetoxy-N-((R)-1 -(1 -benzyl-4-(2,5- difluorophenyl)-1 H-imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1-carboxylate (50 mg, 0.071 mmol) in DMF 5 mL was added DIPEA (0.374 mL, 2.140 mmol) followed by bis(4-nitrophenyl) carbonate (50 mg, 0.164 mmol). Reaction mixture was stirred at room temperature for 16 h. Upon completion by LC/MS (uplc), was added tert-Butyl 3-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)ethyl)amino)propanoate (Linker 2, TFA salt) (54.6 mg, 0.143 mmol), and the reaction mixture was stirred at room temperature for 16 h. Ethyl acetate and cold water were added, organic layer was washed twice with brine, dried with Na₂S0₄, filtered and solvent was removed in vacuo. Absorbed onto Isolute. The desired product was obtained after purification by column chromatography (4 g silica gel, 0 to 100 % EtOAc in Heptane, 40 mg, 0.040 mmol, 56%) as a colorless oil. LC/MS (Method A): MH+ 995.8, 1 .47 min.

3-((((R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutoxy)carbonyl)(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)ethyl)amino)propanoic acid

(3R,4R)-tert-butyl 3-((R)-2-((S)-2-acetoxypropanoyl)-3-(1-benzyl-4-(2,5-difluorophenyl)- 1 H-imidazol-2-yl)-9-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)-4,4, 14,14-tetramethyl- 8,12-dioxo-7,13-dioxa-2,9-diazapentadecyl)-4-fluoropyrrolidine-1-carboxylate (40 mg, 0.040 mmol) was dissolved in a mixture of acetonitrile (1 mL) and water (1 mL). TFA (0.155 mL, 2.010 mmol) was added and reaction mixture was stirred at 60°C for three days.

The desired product was obtained after purification by reverse phase column chromatography (13 mg, 35%, as a TFA salt). LC/MS (Method B): MH+ 797.4, 3.43 min. A mixture of rotamers by ¹ H-NMR at room temperature, some peaks are hidden under water. ¹ H-NMR (DMSO, 600 MHz): δ 12.27 (1 H, br s), 8.98 (1 H, br s), 8.63 (1 H, br s), 7.96 (1 H, s), 7.76 (1 H, s), 7.45-7.30 (6H, m), 7.16-7.09 (1 H, m), 6.97 and 6.96 (2H, two singlets, rotamers), 5.83 (1 H, s), 5.45-5.37 (1 H, m), 5.26 (1 H, d, 54 Hz), 5.09-5.03 (1 H, m), 4.62-4.57 (1 H, m), 4.04-3.90 (2H, m), 3.82-3.66 (2H, m), 2.46-2.35 (3H, m), 2.00-1.85 (2H, m), 1.55-1.20 (5H, m), 1.03 and 0.96 (3H, two singlets, rotamers), 0.67 and 0.63 (3H, two singlets, rotamers).

(R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3-dimethylbutyl (2-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethyl)carbamate

(3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)-4-(((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)carbamoyl)oxy)-2,2- dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (50 mg, 0.073 mmol) was dissolved in a mixture of acetonitrile (1 mL) and water (0.5 mL). TFA (0.222 mL, 2.88 mmol) was added and reaction mixture was stirred at 60C for 20 h. The desired product was obtained after purification by reverse phase column chromatography (17 mg, 34%, as a TFA salt). LC/MS (Method B): MH+ 725.4, 3.59 min. ¹H-NMR (DMSO, 600 MHz): δ 9.01 (1 H, br s), 8.68 (1 H, br s), 7.96 (1 H, s), 7.76 (1 H, s), 7.45-7.30 (6H, m), 7.15-7.03 (2H, m), 6.99 (2H, s), 5.84 (1 H, s), 5.40 (1 H, d, 15 Hz), 5.25 (1 H, d, 54 Hz), 5.06 (1 H, d, 15 Hz), 4.62-4.56 (1 H, m), 4.05-3.65 (4H, m), 3.45-3.40 (2H, m), 3.37-3.15 (2H, m), 3.12-3.05 (2H, m), 2.42-2.33 (1 H, m), 2.00-1.85 (2H, m), 1.60-1.50 (1 H, m), 1.35 (3H, d, 5.9 Hz), 1.32-1.24 (1 H, m), 0.93 (3H, s), 0.73 (3H, s).

(R)-4-(4-(2.5-difluorophenyl)-1-(3-hvdroxybenzyl)-1 H-imidazol-2-yl)-4-gS)-N-

(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hvdroxypropanamido)-3,3-dimethylbutyl

(2-(2,5-dioxo-2,5-dihvdro-1 H-pyrrol-1-yl)ethyl)carbamate

Step 1 (a) - Activation : A stirring solution of (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)- 1 -(4-(2,5-difluorophenyl)-1 -(3-(methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-4- hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4-fluoropyrrolidine-1-carboxylate

(128mg) in DMF (4ml_) was added DIPEA (304μΙ) and bis(4-nitrophenyl)carbonate (88mg). The mixture was stirred at RT for 2 hours - by which time LC-MS showed that the reaction was complete with conversion to desired intermediate product.

Step 1 (b) - Aminolvsis : To the crude mixture above was added N-(2- Aminoethyl)maleimide trifluoroacetate (98mg). The RM stirred at 50°C for a further 2 hours - by which time LC-MS showed conversion to desired intermediate product. The RM was transferred to a separating funnel and de-ionised water (40mL) and ethyl acetate (40mL) added. After extraction the aqueous layer was re-extracted with ethyl acetate (40ml) and the combined organics were then washed with sat. brine (80mL), dried over MgSC>4, filtered through No. 1 filter paper and the filtrate concentrated in vacuo to give a yellow oil. Crude Yield : 270mg This was carried forward directly to the next step.

Step 2 : BOC & MOM & Acyl Deprotection: The crude product from step 1 (b) was dissolved in acetonitrile (1 ml) and 6M HCI (1 mL) added. The RM was stired at 60°C for 3 hours - by which time LC-MS showed that the reaction had almost run to completion with deprotection to desired final product. The RM was filtered through a 0.2pm PTFE syringe filter and the resultant clear solution was purified by reverse phase preparative-scale HPLC, Method C. The product containing fraction was lyophilised overnight to give a white fluffy powder. Yield : 24.7mg

UPLC-MS: Rt = 0.85 min; MS m/z [M+H]⁺ 741.3; Method A.

UPLC-MS: Rt = 3.30 min; MS m/z [M+H]⁺ 741.3; Method B

1 H-NMR (HSQC) consistent with target structure.

(R)-4-(4-(2.5-difluorophenyl)-1-(3-fluoro-5-hvdroxybenzyl)-1H-imidazol-2-yl)-4- S)-N-

(2-(2,5-dioxo-2,5-dihvdro-1 H-pyrrol-1-yl)ethyl)carbamate

Step 1 : A stirring solution of Triphosgene (1 19mg) in anhydrous DCM (5ml_) was cooled to 0°C and placed under an argon atmosphere. A solution of (3R,4R)-tert-butyl 3-(((S)-2- acetoxy-N-((R)-1 -(4-(2,5-difluorophenyl)-1 -(3-fluoro-5-(methoxymethoxy)benzyl)-1 H- imidazol-2-yl)-4-hydroxy-2,2-dimethylbutyl)propanamido)methyl)-4- fluoropyrrolidine-1 -carboxylate

(80mg) and DIPEA (0.258ml) in DCM (2ml) was added dropwise and the RM allowed to warm to RT. Stirring was continued at RT for 2 hours.

Step 2 : The crude RM above was cooled to 0°C and a solution of N-(2- Aminoethyl)maleimide trifluoroacetate (150mg) and DIPEA (0.258ml) in DCM (3ml_) added dropwise. The RM was allowed to warm to RT and stirring continued for 10 minutes. The temperature was then raised to 50°C and stirring continued for 1 hour - by which time LC-MS showed conversion to desired intermediate product. The RM was concentrated under high vacuum to give a dark orange oil. This was taken forward directly to the next step

Step 3 : BOC, MOM & Acyl Deprotection: The crude product from step 2 was dissolved in acetonitrile (2ml) and 6M HCI (1.6mL) added. The RM was stired at 60°C for 2 Hours - by which time LC-MS showed that the reaction had run to completion with deprotection to the final product. The RM was filtered through a 0.2μιη PTFE syringe filter and the resultant solution split into 2x2ml_ batches and purified by reverse phase preparative- scale HPLC, Method C.

Product containing fractions were combined and then lyophilised overnight to give a white fluffy powder Yield : 34.3mg

UPLC-MS: Rt = 0.74 min; MS m/z [M-H]^" 757.3; Method A.

UPLC-MS: Rt = 2.94 min; MS m/z [M+H]⁺ 759.2; Method B

¹ H NMR (600 MHz, DMSO-c ₆) δ 10.10 (s, 1 H), 9.03 (s, 1 H), 8.70 (s, 1 H), 7.93 (s, 1 H), 7.81 - 7.73 (m, 1 H), 7.41 - 7.29 (m, 1 H), 7.19 - 7.10 (m, 1 H), 7.06 (t, J = 6.2 Hz, 1 H), 6.98 (s, 2H), 6.70 - 6.58 (m, 2H), 6.53 (d, J = 10.5 Hz, 1 H), 5.79 (s, 1 H), 5.37 - 5.21 (m, 2H), 4.95 (d, J = 15.6 Hz, 1 H), 4.60 (q, J = 6.3 Hz, 1 H), 4.03 (dd, J = 16.1 , 4.4 Hz, 1 H), 3.89 (dd, J = 15.9, 7.4 Hz, 1 H), 3.82 (q, J = 8.3 Hz, 2H), 3.65 - 3.37 (m, 2H), 3.37 - 3.17 (m, 2H), 3.08 (q, J = 5.9 Hz, 2H), 2.47 - 2.33 (m, 1 H), 2.07 - 1 .84 (m, 2H), 1 .69 (dt, J = 14.2, 7.0 Hz, 1 H), 1 .35 (d, J = 6.2 Hz, 4H), 0.95 (s, 3H), 0.77 (s, 3H).

(R)-4-(4-(2.5-difluorophenyl)-1 -(3-hvdroxybenzyl)-1 H-imidazol-2-yl)-N-(2-(2.5-dioxo-

2.5-dihvdro-1 H-pyrrol-1 -yl)ethyl)-4-((S)-N-g(3S.4R)-4-fluoropyrrolidin-3-yl)methyl)-2- hvdroxypropanamido)-3,3-dimethylbutanamide

Step 1 : To a solution of (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1 -(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1 -(3- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid (80mg) and DIPEA (189μΙ_) in anhydrous DMF (2ml_) was added HATU (82mg). The RM was stired at RT for 10 minutes and then N-(2-Aminoethyl)maleimide trifluoroacetate (92mg) added. The RM was stirred at RT over the weekend - by which time LC-MS showed that the reaction was complete. The RM was partioned between dichloromethane (40ml_) and de-ionised water (40ml_).The mixture was transferred to a separating funnel and, after extraction, the aqueous phase was re-extracted with dichloromethane (40ml). The combined organics were then washed with de-ionised water (40ml), sat. brine (50ml), dried over MgSC>4, filtered through No. 1 filter paper and concentrated in vacuo to give a brown wax-like oily substance. Crude yield : 251 mg. Product still contained some DMF but was carried forward directly to next step:

Step 2 : BOC, MOM & Acyl Deprotection: To the crude product from step 1 was added acetonitrile (4ml) and 6M HCI (1.2mL). The RM was stired at 60°C for 2 Hours - by which time LC-MS showed that the reaction had run to completion with deprotection to give desired final product. The RM was filtered through a 0.2μιη PTFE syringe filter and the resultant clear solution was split into 2x2.6ml_ batches and purified by reverse phase preparative-scale HPLC, Method C. The product containing fractions were combined and lyophilised overnight to give a beige/cream-coloured fluffy powder. Yield : 7mg

UPLC-MS: Rt = 2.72 min; MS m/z [M+H]⁺ 71 1.3; Method B

1 H-NMR (HSQC) consistent with target structure.

(R)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5-hvdroxybenzyl)-1 H-imidazol-2-yl)-N-(2- (2.5-dioxo-2.5-dihvdro-1 H-pyrrol-1-yl)ethyl)-4-((S)-N-g(3S.4R)-4-fluoropyrrolidin-3- yl)methyl)-2-hvdroxypropanamido)-3,3-dimethylbutanamide

Step 1 : To a solution of (R)-4-((S)-2-acetoxy-N-(((3R,4R)-1-(tert-butoxycarbonyl)-4- fluoropyrrolidin-3-yl)methyl)propanamido)-4-(4-(2,5-difluorophenyl)-1-(3-fluoro-5- (methoxymethoxy)benzyl)-1 H-imidazol-2-yl)-3,3-dimethylbutanoic acid (144mg) and DIPEA (450μΙ_) in anhydrous DCM (2ml_) was added HATU (147mg). The RM was stired at RT for 10 minutes and then N-(2-Aminoethyl)maleimide trifluoroacetate (164mg) added. The RM was stirred at RT for 2 hours - by which time LC-MS showed that the reaction to give intermediate product was complete. The RM was concentrated in vacuo and the residue dissoved in acetonitrile (3mL). The solution was filtered through a 0.2μιη PTFE syringe filter and split into 2x1.5mL batches. These were purified by reverse phase preparative-scale HPLC, Method C. The product containing fractions were combined and concentrated in vacuo to give a white powder. Yield : 43mg

Step 2 : BOC, MOM & Acyl Deprotection: The product from step 1 was dissolved in acetonitrile (2ml) and 6M HCI (1.OmL) added. The RM was stired at 60°C for 2 Hours - by which time LC-MS showed that the reaction had run to virtual completion with deprotection to the final product. The RM was filtered through a 0.2μιη PTFE syringe filter and the resultant solution split into 2x2ml_ batches and purified by reverse phase preparative-scale HPLC, Method C.

The product containing fractions were combined and then lyophilised overnight to give a white fluffy powder. Yield : 23.2mg

UPLC-MS: Rt = 2.56 min; MS m/z [M-H]^" 727.4; Method B

1 H-NMR (HSQC) consistent with target structure.

Cysteine Metabolites:

General Procedure for the Synthesis of Cvsteine-Metabolites:

To a solution of Boc-Linker-Payload (0.1 mmol, 1 eq)) in Acetonitrile (1 mL) were added L-cysteine (1 mmol, 10 eq) and water (0.1 mL). The reaction mixture was stirred for 1 h at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude (Boc-Cys-Metabolite) was used as is in the next step.

To the solution of the previous step, TFA (150 mmol) was added and the reaction mixture stirred at room temperature. Upon completion of the reaction by LC/MS (uplc, Method A), the crude was purified by reverse phase column chromatography (PrepLC Method C or D), gradient MeCN (+0.1 % TFA) in H₂0 (+0.1 % TFA). The product was isolated as the TFA salt. (2R)-2-amino-3-((1 -((1 R,5S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8-dioxo-1-(tetrahydro-2H-pyran- 4-yl)-7,12-dioxa-2,4,9-triazatetradecan-14-yl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid.

Boc-Cys-metabolite : LC/MS (uplc): MH+ 1017.6, 1.07 min. (Method A).

21 mg, 0.018 mmol, 16%. ¹H-NMR (DMSO, 600 MHz): δ 9.17-8.59 (1 H, m), 8.81-8.59 (1 H, m), 8.58-8.31 (3H, m), 7.83-7.69 (2H, m), 7.45-7.36 (2H, m), 7.35-7.21 (4H, m), 7.14-6.98 (2H, m), 6.43-6.23 (1 H, m), 5.38-5.28 (2H, m), 5.27-5.10 (2H, m), 4.31-4.21 (1 H, m), 4.15- 4.03 (1 H, m), 4.00-3.80 (4H, m), 3.78-3.68 (1 H, m), 3.27-3.15 (5H, m), 3.14-3.02 (4H, m), 2.65-2.47 (4H, m), 2.33-2.15 (1 H, m), 2.07-1.90 (1 H, m), 1.46-1.32 (1 H, m), 1.28-1.15 (2H, m) 1.14-1.02 (3H, m), 0.79-0.58 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 917.6, 0.80 min. (Method A).

(3R)-6-((1R,5S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8,16-trioxo-1 -(tetrahydro-2H-pyran-4-yl)- 7,12-dioxa-2,4,9,15-tetraazaheptadecan-17-yl)-5-oxothiomorpholine-3-carboxylic acid.

4 mg, 0.0037 mmol, 99 %. ¹H-NMR (DMSO, 600 MHz): δ -NMR (DMSO, 600 MHz): . yl- 4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-(((3S^R)-4-fluoropyrrolidin-3-yl)methyl)-5- methyl-3,8,16-trioxo-42-6.21 (1 H, m), 5.40-5.09 (4H, m), 4.38-4.26 (1 H, m), 4.06-3.80 (5H, m), 3.80-3.68 (1 H, m), 3.68-3.55 (2H, m), 3.26-3.07 (8H, m), 3.05-2.91 (1 H, m), 2.80-2.69 (1 H, m), 2.66-2.54 (1 H, m), 2.36-2.15 (1 H, m), 2.09-1.91 (1 H, m), 1.47-1.35 (1 H, m), 1.32- 1.15 (2H, m), 1.14-1.04 (3H, m), 0.78-0.59 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): M+ 917.4, 084 min.

(2R)-2-amino-3-((1 -((1 -((S)-2-(3-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)ureido)propyl)-1 H-1,2,3-triazol-4-yl)methyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoic acid.

Boc-Cys-metabolite: LC/MS (uplc): MH+ 967.5, 1.06 min. (Method A). 10 mg, 0.009 mmol, 62%. ¹H-NMR (DMSO, 600 MHz): δ 9.20-8.90 (1 H, m), 8.69-8.23 (4H, m), 8.08-7.97 (1 H, m), 7.83-7.70 (2H, m), 7.41-7.36 (2H, m), 7.35-7.29 (2H, m), 7.26-7.22 (2H, m), 7.14-7.08 (1 H, m), 6.58-6.31 (1 H, m), 5.39-5.07 (4H, m), 4.72-4.58 (2H, m), 4.49- 4.34, (2H, m), 4.33-4.06 (4H, m), 3.92-3.81 (2H, m), 3.79-3.67 (2H, m), 3.1 1-2.98 (1 H, m), 2.70-2.43 (4H, m), 2.38-2.18 (1 H, m), 2.06-1.80 (1 H, m), 1.41 -1.29 (1 H, m), 1.28-1.19 (1 H, m), 1.18-1.02 (4H, m), 0.75-0.55 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 867.5, 0.78 min. (Method A).

(2R)-2-amino-3-((1 -(4-(((S)-2-(3-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)-3-(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)ureido)propyl)amino)-4-oxo-2-sulfobutyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoic acid.

Boc-cys-metabolites (mixture of diastereoisomers): LC/MS (uplc): MH+ 1051.5, 0.97 min. (Method A).

The mixture of diastereoisomers was separated after Boc-deprotection using reverse phase chromatography (PrepLC Method A).

Isomer A, 27 mg, 0.024 mmol, 8%. ¹H-NMR (DMSO, 600 MHz): δ 9.12-8.96 (1 H, m), 8.96-8.78 (1 H, m), 8.48-8.24 (3H, m), 7.97-7.84 (1 H, m), 7.81 -7.65 (2H, m), 7.48-7.37 (2H, m), 7.36-7.24 (4H, m), 7.17-7.01 (1 H, m), 6.18-6.01 (1 H, m), 5.49-5.16 (4H, m), 4.34-4.20 (1 H, m), 4.15-4.05 (1 H, m), 3.96-3.66 (6H, m), 2.87-2.64 (4H, m), 2.61-2.39 (3H, m), , 2.13-1.97 (1 H, m), 1.93-1.58 (2H, m), 1.50-1.36 (1 H, m), 1.35-1.20 (1 H, m, 1.19-0.93 (4H, m), 0.71-0.52 (1 H, m). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 951.5, 0.79 min. (Method A).

Isomer B, 28 mg, 0.025 mmol, 8%. ¹H-NMR (DMSO, 600 MHz): δ 9.14-8.96 (1 H, m), 8.86-8.61 (1 H, m), 8.51-8.24 (3H, m), 8.1 1-7.89 (1 H, m), 7.80-7.65 (2H, m), 7.49-7.38 (2H, m), 7.37-7.23 (4H, m), 7.16-7.01 (1 H, m), 6.01-5.90 (1 H, m), 5.49-5.17 (4H, m), 4.35-4.19 (1 H, m), 4.16-3.96 (2H, m), 3.92-3.66 (6H, m), 3.1 1-2.98 (1 H, m), 2.89-2.77 (1 H, m), 2.75- 2.59 (1 H, m), 2.58-2.44 (4H, m), 2.10-1.93 (1 H, m), 1.72-1.56 (1 H, m), 1.41-1.24 (2H, m), 1.21-1.09 (1 H, m), 1.08-0.95 (3H, m), 0.75-0.53 (1 H). Missing signals hidden under the solvent peak. LC/MS (uplc): MH+ 951.5, 0.81 min. (Method A).

(2R)-2-amino-3-((1 -((1 R,5S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-5-methyl-3,8-dioxo-1-(tetrahydro-2H-pyran- 4-yl)-7-oxa-2,4,9-triazapentadecan-15-yl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid.

Boc-Cys-Metabolite: LC/MS (uplc): M+ 1029.7, 1.12 min (Method A).

Cys-Metabolite: LC/MS (uplc): M+ 929.6, 0.83 min (Method A).

(2R)-2-amino-3-((1 -((1 R,4S)-1 -(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-4-methyl-3,6-dioxo-1-(tetrahydro-2H-pyran- 4-yl)-5,10-dioxa-2,7-diazadodecan-12-yl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid.

Boc-Cys-Metabolite: LC/MS (uplc): M+ 988.5, 1.10 min (Method A).

Cys-Metabolite: LC/MS (uplc): M+ 888.5, 0.79 min (Method A).

(3R)-6-((1R,4S)-1-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-2-(((3S,4R)-4- fluoropyrrolidin-3-yl)methyl)-4-methyl-3,6,14-trioxo-1 -(tetrahydro-2H-pyran-4-yl)- 5,10-dioxa-2,7,13-triazapentadecan-15-yl)-5-oxothiomorpholine-3-carboxylic acid.

Cys-Metabolite: LC/MS (uplc): M+ 888.5, 0.83 min (Method A).

(2R)-2-amino-3-((1 -(6-(((((S)-1 -(((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)amino)- 1-oxopropan-2-yl)oxy)carbonyl)amino)hexyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoic acid.

Boc-Cys-Metabolite: LC/MS (uplc): M+ 1000.5, 1.14 min (Method A).

Cys-Metabolite: LC/MS (uplc): M+ 900.4, 0.83 min (Method A).

(2R)-2-amino-3-((1 -(3-((S)-2-(((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(tetrahydro-2H-pyran-4-yl)methyl)(((3S,4R)-4-fluoropyrrolidin-3- yl)methyl)carbamoyl)morpholino)-3-oxopropyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoic acid

Step 1 : To (3R,4R)-tert-butyl 3-(((S)-N-((R)-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol- 2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-4-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)propanoyl)morpholine-2-carboxamido)methyl)-4-fluoropyrrolidine-1-carboxylate (25 mg, 0.029 mmol) in Acetonitrile (Volume: 1.5 ml) and Water (Volume: 0.500 ml), L-Cysteine (5.35 mg, 0.044 mmol) was added and RM stirred at RT for 2 h. Step 2: HCI (25% aq.) (0.179 ml, 1.472 mmol) was added and RM stirred at RT for 2 h, another HCI (25% aq.) (0.179 ml, 1.472 mmol) were added and RM was stirred at RT for 4 h. Concentrated.

Purified by reverse phase chromatography to afford colorless solid double TFA salt (16mg, 0.015 mmol, 50 % yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 870.2; Rt 2.73 min.

(2R)-2-amino-3-((1 -(2-((R)-4-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)- N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanamido)ethyl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid

Step 1 : (3R,4R)-tert-butyl 3-(((S)-N-((R)-1 -(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)-4-((2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)ethyl)amino)-2,2-dimethyl-4-oxobutyl)-2- hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (30 mg, 0.038 mmol) in Acetonitrile (Volume: 1.5 ml) and Water (Volume: 0.500 ml), L-Cysteine (13.7 mg, 0.1 13 mmol) was added and RM stirred at RT for 16 h.

Step 2: TFA (0.145 ml, 1.887 mmol) was added and RM stirred at RT for 60 h.

Concentrated.

Purified by reverse phase chromatography to afford colorless solid double TFA salt (37mg, 0.035 mmol, 93 % yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 816.3; Rt 2.37 min. (2R)-2-amino-3-((1 -(2-(2-((R)-4-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H-imidazol-2-yl)-4- ((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanamido)ethoxy)ethyl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid

Step 1 : (3R,4R)-tert-butyl 3-(((S)-N-((R)-1 -(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)-4-((2-(2-(2,5-dioxo-2,5-dihydro-1 H^yrrol-1-yl)ethoxy)ethyl)amino)-2,2-dimethyl-4- oxobutyl)-2-hydroxypropanamido)methyl)-4-fluoropyrrolidine-1-carboxylate (54 mg, 0.055 mmol, 85% pure) in Acetonitrile (Volume: 1.5 ml) and Water (Volume: 0.500 ml), L- Cysteine (19.9 mg, 0.164 mmol) was added and RM stirred at RT for 16 h.

Step 2: TFA (0.21 1 ml, 2.74 mmol) was added and RM stirred at 60^°C for 4 h.

Concentrated.

Purified by reverse phase chromatography to afford colorless solid double TFA salt (48mg, 0.044 mmol, 81 % yield, 99% pure).

LC/MS (Method B): [M+H]⁺ 860.5; Rt 2.50 min.

(2S)-2-amino-3-((1-((S)-2-((((R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4- ((S)-N-(((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutoxy)carbonyl)amino)-2-carboxyethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoic acid

1 1 mg of colorless powder were obtained as a TFA salt (1 1 mg, 9.84 μιηοΙ, 13.2%yield, 100% pure).

LC/MS (Method B): [M+H]⁺ 890.3 ; Rt 2.54/2.63 min (two diastereomers).

Cysteine metabolites of Compounds 5A, 5D and 5E were prepared and all were highly potent inhibitors of Eg5, with IC-50s of < 0.5 nM, 0.5 nM, and 0.6 nM, respectively.

General protocol 2 for Catabolite Synthesis

To a solution of (L)-Cysteine (10 equiv.) in water (0.1 M) was added the Boc-protected Linker-payload (1.0 equiv.) in DMF (0.1 M) and the reaction was stirred at room temperature for 1 h. The reaction was with ethyl acetate. The organic extracts were combined, dried over Na₂S0₄, filtered and concentrated to dryness. The residue was dissolved in DCM (0.1 M) and trifluoroacetic acid (50 equiv.) was added. The reaction was stirred at RT for 2h before it was concentrated to dryness. Purification of the crude product by chromatography on silica elutuing with 0 - 100% methanol in DCM afforded the title compound.. (2R)-2-amino-3-((1 -(2-(((((3S,4S)-4-(((S)-N-((R)-(1 -benzyl-4-(2,5-dif luorophenyl)-1 H- imidazol-2-yl)(tetrahydro-2H-pyran-4-yl)methyl)-2- hydroxypropanamido)methyl)pyrrolidin-3-yl)oxy)carbonyl)amino)ethyl)-2,5- dioxopyrrolidin-3-yl)thio)propanoic acid

49% yield; UPLC-MS: Rt = 1 .66 and 1 .67 mins; MS m/z [M+H]⁺ 842.3; Method A. ¹H- NMR (DMSO, 400 MHz, mixture of rotamers and diastereoisomers): δ 7.99-6.77 (12H, m), 5.64-4.99 (3H, m), 4.87-4.85 (1 H, m), 4.53-4.51 (1 H, m), 4.03-2.05 (25H, m), 1 .51 - 0.57 (7H, m).

Rearranged Cysteine Metabolites:

(3R)-6-(2-((2-((R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanamido)ethyl)amino)-2-oxoethyl)-5-oxothiomorpholine-3-carboxylic acid

(2R)-2-amino-3-((1-(2-((R)-4-(1-benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2-yl)-4-((S)-N- (((3S,4R)-4-fluoropyrrolidin-3-yl)methyl)-2-hydroxypropanamido)-3,3- dimethylbutanamido)ethyl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid (17 mg, 0.016 mmol) was dissolved in Acetonitrile (Volume: 0.5 ml_) and 1.5 ml_ of PBS buffer (pH7.2) were added. RM stirred at RT for 10 days. Purified by reverse phase chromatography to afford colorless solid double TFA salt (9mg, 0.016 mmol, 57 % yield, 95% pure).

LC/MS (Method B): [M+H]⁺ 816.5; Rt 2.54 min.

Synthesis of ADCs

ADCs were obtained and characterized as described in the following general conjugation procedures.

General Methods for Conjugation of Linker-Payload (L-P) with Antigen Binding Moiety

Payloads were conjugated to antigen binding moieties (e.g., lgG1 kappa or lambda of an antibody) at partially reduced hinge and inter-chains disulfides in a 2-step process. The antibody at a concentration of 5-10 mg/ml in PBS containing 2 mM EDTA, was first partially reduced for 1 to 1.5 hour at 37 °C with 50 mM mercaptoethylamine (added as a solid). After desalting and addition of 1 % w/v PS-20 detergent, the partially reduced antibody (1-2 mg/ml) was reacted overnight at 4 °C with an amount of 0.5-1 mg of the Linker-Payload compound, dissolved at 10 mg/ml in DMSO or other suitable solvents, per 10 mg antibody. The antibody-drug conjugate is purified by Protein A chromatography. After base-line washing with PBS, the conjugate is eluted with 50 mM citrate, pH 2.7, 140 mM NaCI, neutralized and sterile filtered. The ADCs described in Tables 5-6 were prepared according to this procedure. This procedure yields an average drug loading of 4-6 molecules of payload per antibody: specific examples of the products of this general procedure are shown in Tables 5-6, which identifies the linking group-payload moiety attached to an antibody having the sequence of trastuzumab, and provides measured DAR values and %aggregation data for each conjugate. Biological data for selected conjugates from Tables 5-6 are provided in the following section and associated Figures.

A different process was used for antibodies where new cysteine residues were introduced by protein engineering as conjugation sites. In order to reduce all native disulfide bonds and the disulfide bound between the cysteine or GSH adducts of the engineered cysteine residue, freshly prepared DTT was added to previously purified Cys mutants of the antibody, to a final concentration of 20 mM. After incubation with DTT at 37°C for 1 hour, the mixtures were dialyzed at 4°C against PBS for three days with daily buffer exchange to remove DTT and re-oxidize the native disulfide bonds. An alternative method is to remove the reducing reagents through a desalting column, Sephadex G-25. Once the protein is fully reduced, 1 mM oxidized ascorbate (dehydro-ascorbic acid) is added to the desalted samples and the re-oxidation incubations are carried out for 20 hours. Reoxidation restores intra-chain disulfides, while dialysis allows cysteines and glutathiones connected to the newly-introduced cysteine(s) to dialyze away. (Both of these methods produce similar results, but attempts to follow the re-oxidation protocols previously described in the literature using CuS0₄ resulted in protein precipitation. All examples herein use the dialysis protocol described above.

Reoxidized antibodies were conjugated with Compound 223 (5B) by incubating 5 mg/ml antibody with 0.35 mM of the maleimide compound for 1 hour in 50 mM sodium phosphate buffer (pH 7.2). The completeness of the reaction was monitored by RP- HPLC and a DAR of 3-4 was typically obtained. For cKitA, DAR for a sample made by this process was 3.2. The engineered antibodies gave slightly higher DAR under the same condition, with DAR = 3.9 for the cKitB— 5B conjugate, and DAR = 4.0 for the cKitC— 5B conjugate. DAR measurements were further verified by MS.

The following ADCs were prepared by the above procedures using an anti-Her2 antibody like trastuzumab as the antibody (referred to herein as "TBS"), demonstrating the generality and efficacy of the Eg5 inhibitors of Formula II as payload compounds. Characterization data (DAR and % aggregation) for numerous immunoconjugates with Ab = TBS are presented in Table 7; structures for ADC No. 1 10 to ADC no. 133 are shown here.

ADC-110.

ADC-111.

ADC-112.

ADC-115.

ADC-117.

ADC-119.

ADC-120.

ADC-123.

ADC-126.

ADC-129.

ADC-132.

Characterization of Antibody-Drug Conjugates

The immunoconjugates prepared as described above were characterized by LC/MS, as illustrated in Figure 1 for one immunoconjugate. Conjugation typically provides a mixture of conjugates that differ in the number of copies of the Linker-Payload moiety bound to the antibody. Mass spectral analysis demonstrates that Linker-Payload groups are attached to light chains and/or heavy chains in each of the conjugates in Table 5-6. The conjugates were characterized in terms of average drug loading for a sample (DAR, drug to antibody ratio) and aggregation (expressed in %). The DAR value is extrapolated from LC-MS data for reduced and deglycosylated samples. As illustrated in Figure 1 for the conjugate of trastuzumab with Compound No. 223 (TBS- Cmpd 223), LC/MS allows quantitation of the average number of molecules of payload (drug) attached to an antibody in an ADC. HPLC separates the antibody into light and heavy chains, and separates the heavy chain (HC) and light chain (LC) according to the number of Linker-Payload groups per chain. Mass spectral data enables identification of the component species in the mixture, e.g., LC, LC+1 , LC+2, HC, HC+1 , HC+2, etc. From the average loading on the LC and HC chains, the average drug to antibody ratio (DAR) can be calculated for an ADC (see Fig. 1 B).. The DAR for a given conjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains. In this example, the DAR is 5.8.

Some conjugates were found to form aggregates, and conjugates described herein were also characterized by the extent of aggregation. Aggregation was measured by analytical size exclusion chromatography (Superdex 200 5/150 GL run in PBS). It was observed that the extent of aggregation depends on the Linker-Payload and the DAR. Conjugates having a lower percent aggregation may be more useful for some purposes, thus immunoconjugates that exhibit less than 50% aggregation, and preferably less than 20% aggregation, may be preferred. The data also demonstrate that the extent of aggregation for a given payload can be manipulated by selection of linking group and DAR. Table 7 also indicates whether the linking group for each conjugate is stable or cleavable.

Table 7. Characterization of Immunoconjugates.

ADC Cmpd # of

Linker Type Aggregation % DAR No. Linker+Payload

7 207 cleavable 44.4 2.7

8 216 stable 1.4 3.2

9 217 cleavable 84.9 6

10 218 stable 47 5

1 1 220 cleavable 4 4

12 220 proteolysis 4.7 5

13 223 stable 0.7 5.8

14 224 stable 1.5 2.8

15 225 stable 3.5 3.7

16 226 cleavable 20.4 7.4

17 227 stable 55 5.8

18 228 cleavable 58.4 3

19 229 cleavable 74.6 4.7

20 230 cleavable 63.5 5.2

21 231 cleavable 39.7 4.7

22 232 cleavable 84.5 6

23 233 cleavable 66.8 3.1

24 234 cleavable 54.2 1.2

25 235 cleavable 60.8 2.2

26 236 cleavable 33.5 2.6

27 237 cleavable 12.1 3.4

28 238 cleavable 63.6 3.6

29 239 cleavable 2.9 2.8

30 240 stable 2.5 3.9

31 241 stable 10.6 5.8

32 243 stable 1.4 5.2

33 244 cleavable 29.8 6.3

34 247 stable 50.5 4.4

35 248 cleavable 44.3 3

36 249 cleavable 40 2.6

37 249 cleavable 68 5.6

38 250 cleavable 67.2 5.8 ADC Cmpd # of

Linker Type Aggregation % DAR No. Linker+Payload

39 254 cleavable 68.5 3.2

40 256 stable 9.3 4

41 257 cleavable 23.8 4.5

42 258 stable 2.4 6

43 259 stable 7.7 6.1

44 260 stable 41 4.8

45 261 stable 22 4.8

46 264 cleavable 74.1 4.7

47 265 stable 65.2 5.1

48 266 stable 63.8 4.4

48 267 stable 35.3 5.2

50 268 cleavable 40 2.8

51 269 cleavable 56.5 5.6

52 270 cleavable 47.4 5.2

53 273 cleavable 25.7 2.7

54 274 cleavable 2.5 3.1

55 276 cleavable 1 1.5 3.8

56 278 stable 45.1 4.1

57 279 stable 57 5

58 280 stable 59.4 5.3

59 281 stable 71 5.2

60 282 proteolysis 71.7 6.4

61 284 cleavable 46.1 2

62 285 cleavable 48.7 2

63 289 stable 53.2 4

64 293 cleavable 49.3 2.9

65 294 cleavable 1 1.4 3.2

66 295 cleavable 64.5 3.6

67 296 cleavable 63.2 3.4

68 297 stable 62.6 4.6

69 298 stable 4.9 6

70 299 cleavable 16.9 6.5 ADC Cmpd # of

Linker Type Aggregation % DAR No. Linker+Payload

71 300 stable 2.6 2.9

72 301 stable 45 5.2

73 302 stable 60 4.5

74 303 cleavable 39.1 3

75 306 cleavable 69.4 2.9

76 307 cleavable 48.5 3.1

77 308 cleavable 47.7 2.8

78 310 cleavable 31.7 5.6

79 31 1 cleavable 44.9 3.4

80 312 cleavable 38 2.8

81 313 cleavable 1 1.6 4.4

82 314 stable 50.1 5.3

83 315 stable 2.2 4.6

84 316 stable 1.2 5

85 317 stable 1.8 6.1

86 318 stable 60 5.1

87 319 cleavable 75 4

88 320 stable 36.3 5.6

89 322 cleavable 56.8 3.8

90 323 stable 60 5

91 324 cleavable 55.8 3

92 326 stable 63.7 4

93 327 stable 43.1 4.6

94 328 stable 53.8 4

95 329 cleavable 1.8 0.2

96 331 cleavable 66.6 3

97 336 cleavable 54 3.4

98 337 cleavable 15.8 4.1

99 338 cleavable 49.7 3.3

100 339 cleavable 1.3 2.8

101 343 stable 19.9 5

102 344 cleavable 13.3 2.1 ADC Cmpd # of

Linker Type Aggregation % DAR No. Linker+Payload

103 345 stable 4 3.5

104 355 stable 30.5 6

105 356 stable 24.2 5.9

106 357 stable 8.2 5.6

107 358 stable 16.8 5.6

108 359 stable 33.8 5.7

109 360 stable 3.1 5.2

1 10 366 Stable 1 1 .6 4.2

1 1 1 367 Stable 2.8 2.4

1 12 368 stable 2.8 2.7

1 13 400 Stable 3.0 4.9

1 14 488 Stable 12.2 4.0

1 15 402 Stable 2.1 4.0

1 16 404 Stable 1 .6 3.7

1 17 406 Stable 2.1 4.0

1 18 407 Stable 1 .9 3.9

1 19 408 Stable 2.3 3.9

120 409 Stable 7.7 2.7

121 410 Stable 4.7 4.2

122 41 1 Stable 2.4 3.5

123 413 Stable 13.9 3.8

124 414 Stable 6.4 2.9

125 415 Stable 2.8 2.7

126 416 Stable 30.5 6.0

127 509 Cleavable 77.4 4.4

128 508 Cleavable 78.1 5.0

129 357 Stable 9.1 4.5

130 356 Stable 24.2 5.9

131 359 Stable 33.8 5.7

132 358 Stable 16.8 5.6

133 500 Stable 5.3 3.5

6H Stable

cmpd 390 2.5 4.4 ADC Cmpd # of

Linker Type Aggregation % DAR

No. Linker+Payload

6F

cmpd 397 Stable 6.3 5.5

6G cmpd 388 Stable 2.4 5.1

6G cmpd 388 Stable 3.4 5.9

61 Stable

61 6.4 2.9

6J

cmpd 398 Stable 2.6 4.5

6K

cmpd 399 Stable 2.4 4.7

6L

449 Stable 4.6 4.7

6Q

483 Stable 2.0 4.3

6R 484 Stable 26.0 4.5

5E Stable

Cmpd 5E 2.6 4.4

5G 316 Stable

1.9 4.1

5H

315 Stable

1.9 3.9

6A Stable

Cmpd 6A 11.6 4.2

6A

Cmpd 6A Stable 9.4 4.0

6B

482 Stable 12.2 4.8

6C Cmpd 6C Stable 7.2 2.7

5C

= 6E

382 stable 3.6 3.9

Structures for certain of these ADCs (ADC-1 10 to ADC-133) are found in the preceding section. Note that ADC-127 and ADC-128 are comparative examples, with payloads outside the scope of Formula (II). Some ADCs appear more than once in the table, representing different preparations of the ADC, which illustrate some variation in %aggregation and DAR. Variations of up to 15-20% in DAR cause little change in observed bioactivity— see Figure 28-29.

While the Examples in Table 7 were conjugated to trastuzumab, several of the linker-payload combinations of the invention have also been conjugated to other antibodies and antigen binding moieties directed at different antigens. Description of other antibodies and summaries of their characterization and activity are provided in the Activity Examples below, and characterization data for numerous of these conjugates are provided below. Conjugates with antibodies directed to other tumor cell antigens were shown to have greater growth inhibiting effects on cells that express high levels of the antigen that is recognized by the antibody in the ADC than on cells lacking that antigen. This demonstrates that ADCs having Eg5 inhibitors of Formula (II) as their payload can be used to target different cell lines or tumor types by selecting an antibody that recognizes an antigen on the targeted cell line, providing evidence that effective conjugates of the invention can utilize antibodies that target other cells and antigens.

The cKitA antibody and anti-gH antigen binding moiety are described in the examples— see Example 5 and Example 8. Activity Example 1. In vitro Anti-Proliferative Activities of Eg5 Inhibitors

Compounds of Formula (II) are structurally similar to known Eg5 inhibitors. Their in vitro activity at the target site was verified by determining IC50's against Eg5. The following table provides IC-50 data for selected compounds of the invention: the lower limit for the assay conditions used was 0.0005 μΜ.

550 0.00084

0.00085

0.0044 0.00775

0.0052

0.0015

Materials and Methods

Cell Lines

To generate a Her2-overexpressing cell line, MDA-MB-231 breast cancer cells were stably transduced with a lentiviral construct (pLenti 6.3 (Invitrogen); driven by a cytomegalovirus enhancer-promoter) encoding a mutant form of the Her2 antigen (NM_004448; codon K753M), lacking kinase activity and therefore non-oncogenic but still recognized by the anti-Her2 antibody. A Her2-overexpressing line, MDA-MB-231 / Her2mutant Clone 16 ("Clone 16"), was isolated by fluorescence-activated cell sorting and selection with blasticidin. In the same manner, the mock-transduced MDA-MB-231 - M40 line (transduced with lentivirus but not expressing exogenous Her2) was isolated. Clone 16 and parental MDA-MB-231 cultures were maintained by passage in RPMI-1640 growth medium, supplemented with 10% (v/v) fetal bovine serum. An alternative model, SK-OV-3ip, was isolated upon serial passage of SK-OV-3 cells in the peritoneal cavities of mice to select for cells that thrive in a rodent host. SK-OV-3ip cells were maintained by passage in McCoy's 5A growth medium, supplemented with 10% (v/v) fetal bovine serum. T47D2 cells are a variant of the T47D breast cancer cell line. Other cell lines expressing high levels of Her2 antigen include the breast cancer line, HCC1954 and the ovarian cancer line, OE-19. The gastric cancer line, NCI-N87, is a moderate expresser of Her2 that provides an additional test of potency. Selectivity for Her2 expression was evaluated using MDA-MB-231 parental and M40 lines, and the Her2-negative breast cancer line, MDA-MB-468. anti-cKit ADCs(cKitA, cKitB and cKitB) were tested on cKit-high NCI-H526 cells (a small cell lung cancer line) and cKit-negative MDA-MB-468 cells. All other cell lines are readily available from a standard vendor. All cell lines were maintained by serial passage in a humidified 37°C incubator charged with an atmosphere of 5% C0₂. Cell proliferation with antibody-drug conjugate treatment

On Day 0, cells were seeded into 96-well clear-bottom, black-wall plates (Costar #3603) at 3000 cells per well in 90 μΙ_ growth medium. On Day 1 , antibody-drug conjugates were diluted into cell growth medium at 10-fold above the final concentrations, starting at 90 μg/mL with three-fold serial dilutions down to 1 .5 ng/mL. Conjugate dilutions (10 \}L/we\\) were then added into the 96-well plates of cells; final concentrations range from 9000 ng/mL down to 0.15 ng/mL. Duplicate or triplicate samples were prepared. Cells were placed in a humidified 37°C incubator charged with an atmosphere of 5% C0₂. On Day 5 or 6, 96-well plates were removed from the incubator and allowed to equilibrate to room temperature. Cell TiterGlo 2 (50 μί/ννβΙΙ; Promega #G7571 ) was added to each well with 10 minutes agitation. Bio-luminescence (indicating relative levels of ATP) was measured using a Wallac MicroBeta luminometer.

Cell proliferation with Eg5 inhibitor treatment

On Day 0, cells were seeded into 96-well clear-bottom, black-wall plates (Costar #3603) at 3000 cells per well in 90 μί growth medium. On Day 1 , Eg5 inhibitors were diluted into cell growth medium at 10-fold above the final concentrations, starting at 1000 nM with three-fold serial dilutions down to 51 pM. Inhibitor dilutions (10 \}L/we\\) were then added into the 96-well plates of cells; final concentrations range from 100 nM down to 5.1 pM. Duplicate or triplicate samples were prepared. Cells were placed in a humidified 37°C incubator charged with an atmosphere of 5% C0₂. On Day 5 or 6, 96- well plates were removed from the incubator and allowed to equilibrate to room temperature. Cell TiterGlo 2 (50 μί/ννβΙΙ; Promega #G7571 ) was added to each well with 10 minutes agitation. Bio-luminescence (indicating relative levels of ATP) was measured using a Wallac MicroBeta luminometer.

Definition and derivation of potency values

Cell TiterGlo 2 data were averaged for replicate samples then normalized to untreated cells. Dose-response curves were derived using a four-parameter logistic model (sigmoidal dose-response model #205) as provided by the XL-Fit software package (IDBS) that is used as an add-on to Microsoft Excel. fit = (A+((B-A)/(1 +((C/x)^AD)))) inv = (C/((((B-A)/(y-A))-1 H1/D))) res = (y-fit)

EC50 = Concentration of test article at which the fitted Cell TiterGlo 2 signal is 50% of the signal generated by untreated cells.

IC50 = Concentration of test article at which the fitted Cell TiterGlo 2 signal is reduced by 50% of the signal differential between untreated cells and the maximal effect of the test article. For example, if the maximal effect is a reduction in signal down to 40% of untreated cells, the IC50 is the concentration at which the fitted dose-response curve reaches 70% of untreated cells. The IC50 is equivalent to parameter "C" in the fitting algorithm provided above.

Results

Cell proliferation in the presence of Eg5 inhibitors was performed as described in the methods above. After 5 or 6 days, cell counts were determined using Cell TiterGlo 2 reagent (FIG. 2), except for FIG. 2(A)-(B), where the incubations were for three days. Data for duplicate samples were averaged, then the averages were normalized to average values of untreated cells or cells treated at the lowest concentration tested. Both normalization methods yielded comparable findings. The comparison of Compound #2 (Table 1 ) and Compound #14 (Table 1 ) in FIG. 2(E)-(F) was performed with single samples per concentration, which were normalized to the lowest concentration tested. Data for cellular activity of selected inhibitors of Formula (II), and for comparative Eg5 inhibitors, are provided in the following table.





These Eg5 inhibitors have anti-proliferative activity across cell lines from many different lineages, indicating that these molecules have broad potential as ADC payloads and as cancer therapeutic agents. The inhibitory concentrations in these assays were generally within a tight range for any given compound. The variations observed in maximal inhibition appear consistent with arrest of the cell cycle at the G2/M transition, followed by apoptosis; the timing of onset of apoptosis varies widely among cell lines, which may explain variances in maximal compound effects.

When comparing among a panel of Eg5 inhibitors, a range of anti-proliferative activity is observed. Note that the rank order of compound potency is generally maintained from one cell line to another. Cellular potency is influenced by many factors, including intrinsic inhibition of Eg5 enzymatic activity and permeability of cellular membranes to the compound. For example, Compound #77 contains a carboxylic acid that will be largely deprotonated at physiological pH, which may explain why this compound is somewhat less potent (FIG. 1 E) than others in this test. A variety of chemical scaffolds are shown to confer strong anti-proliferative activity. For example, FIG. 2(A)-(E) shows examples of Eg5 inhibitors in the t-butyl and THP series (R¹ = t-Butyl or 4-tetrahydropyranyl, respectively), and THP series inhibitors with a core urea (A = NH) or a core amide (A = bond). Examples from each series inhibit proliferation at sub-nanomolar concentrations. Compounds with relatively less potency are also observed: in some cases, the compounds with lower potency were

disproportionately effective as inhibitors of cell proliferation when delivered to cells in the form of antibody-drug conjugates.

FIG. 3(A)-(L) illustrates examples of anti-proliferative activities of specific Eg5 inhibitors across a variety of cancer cell lines derived from different lineages (see Table 8 below). Although the potencies do vary, all cell lines across these lineages are sensitive to the compounds of Formula (II) and (III).

Table 8.

Activity Example 2. In Vitro Anti-Proliferative Activity of Eg5 Inhibitor ADCs

Cell proliferation in the presence of antibody-drug conjugates ("ADCs") with anti- Her2 trastuzumab antibody ("TBS") and Eg5 inhibitors was performed as described in the methods above. After 5 or 6 days, cell counts were determined using Cell TiterGlo 2 reagent. Data for duplicate samples were averaged, then the averages were normalized to average values of untreated cells. FIG. 4A-V shows pairs of dose-response graphs, illustrating the anti-proliferative effects of the ADCs on Her2-high Clone 16 cells vs. Her2- low parental MDA-MB-231 cells. Irrespective of the specific linker chemistry employed, the activity of the ADCs was highly selective for elevated Her2 expression. FIG. 4A-H illustrate the in vitro potencies of ADCs employing linkers that are designed to be cleaved in lysosomes, releasing the unmodified Eg5 inhibitor inside the target cell. Combinations of different payloads (from different structural families) with two different cleavable linkers (containing the dipeptide valine-citrulline or the glycan - glucuronide) were generally anti-proliferative against Clone 16 cells but much less so against parental MDA-MB-231 cells.

FIG. 4G-H illustrate the selective activity of TBS-Cmpd312, which incorporates the carboxylate-containing Eg5 inhibitor Compound #77. The free compound has modest anti-proliferative activity (FIG. 2E), possibly due to poor membrane permeability at extracellular pH (around 7). When released in acidic lysosomes (pH around 5), the carboxylate will be partially protonated, removing a charge and improving membrane permeability, which in turn could explain the activity of the ADC.

It is anticipated that different properties may be obtained through the use of non- cleavable linkers, whereby the intracellular metabolism of the ADC will generate an adduct of payload and linker coupled to one or more amino acids derived from the antibody. In vitro anti-proliferative activities of exemplary ADCs with non-cleavable linkers are illustrated in FIG. 4I-R. This series comprises linkers attached at a region of the Eg5 inhibitors that is known or predicted not to be involved in Eg5 binding. The linkers represented in FIG. 4I-R vary with respect to modes of attachment to the payload and to the antibody cysteine. Additionally, the linkers vary in length and in predicted physical properties such as lipophilicity and conformational flexibility.

All of these ADCs attach to the antibody cysteine via a maleimide group in the linker, except for TBS-Cmpd300 (FIG. 4K, L, O, P), which employs an iodoacetamide group for thiol coupling. The Her2-selective in vitro potency of this ADC demonstrates that the maleimide is not required for anti-proliferative activity.

Non-cleavable linkers attached to the payloads at different sites were employed to produce the ADCs with in vitro activity illustrated in FIG. 4S-V. Again, attachment of the lin linking group ker at a different position on the payload does not prevent achieving Her2-depenent cellular potency, demonstrating that the linking group attachment point on the compounds of Formula (II) can be varied.

To demonstrate that the ADCs can inhibit the proliferation of cells that express Her2 endogenously, selected ADCs with the anti-Her2 antibody were incubated with either HCC1954 (FIG. 5A, B) or SK-OV-3ip (FIG. 5C-E) cells. ADCs with either cleavable or non-cleavable linkers, derived from different linker-payload structural families, inhibited the proliferation of these Her2-high cell lines.

Activity Example 3. In Vivo Efficacy Assessment of Eg5 Inhibitor ADCs

Compounds of the invention conjugated to trastuzumab (TBS) also demonstrated significant activity in xenograft tumor model, which is based on the implantation of a human tumor cell line into immune-deficient nude mice. As described previously

(Sausville and Burger, 2006), studies with such tumor xenograft mice have provided valuable insights into the in vivo efficacy of anti-cancer reagents. Specifically, the in vivo efficacy study was carried out with nu/nu mice that were subcutaneously injected with 5.0 x 10⁶ SK-OV-3ip cells (Yoneda et al., 1998) or HCC1954 cells. These cell lines were chosen based on previous in vitro potency assays revealing their high sensitivity to the aforementioned Eg5 inhibitor ADCs in an antigen dependent manner. After the tumor reached a size of about 200-250 mm³, the Eg5 inhibitor ADCs were intravenously injected in a single dose, at doses from 0.3 mg/kg to 10 mg/kg depending on the experiment, with each treatment group comprising nine mice. After administering the antibody-drug conjugate, the tumor volume was monitored twice weekly. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8th edition, 2001 ).

FIGs 6(A) and 6(B) show efficacy of an ADC having Linker-Payload Compound No. 220 conjugated with trastuzumab (TBS-Compound 220) on HCC1954 breast cancer xenograft tumors in mice. Figure 6(A) shows tumor size changes over a period of about 55 days with a single dose of 1 , 2, or 3 mg/kg of the TBS-Compound 20 conjugate, which contains compound 12 (Table 1 ) as payload. The 1 mg/kg dose shows modest tumor growth reduction compared to a control having no Eg5 inhibitor, while the 2mg/kg and 3 mg/kg doses prevented tumor growth during the test. Figure 3b shows results of 3 mg/kg and 6 mg/kg dosages, which prevented tumor enlargement.

Animals treated with TBS alone (the anti-Her2 antibody containing no Eg5 inhibitor)

(FIG. 6(B)) at 6 mg/kg, corresponding to the amount of antibody in the highest conjugate dose tested, allowed approximately quadrupling of tumor volume over the test period and appear similar to vehicle-only control treatments. Other animals received the Isotype control-Compound 220 conjugate, which is a conjugate with the same payload attached to an antibody that does not target the HCC1954 cells. At similar dosages of the Eg5 inhibitor, the isotype controls displayed slight tumor growth inhibition relative to the TBS control, but were clearly less effective at suppressing tumor growth than the TBS- Compound 220 conjugate that does target the HCC1954 cells, except at a dose of 6 mg/kg where the isotype control showed nearly comparable activity to the TBS- Compound 220.

FIGs 7(A) and 7(B) show similar results in SKOV3ip xenografts. A single dose of a conjugate of compound 220 with TBS was administered to mice at doses of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg and 10 mg/kg, which delivers a dose of from 5 to 96 micrograms/kg of Compound 12. In this xenograft, doses of 3 mg/kg of the TBS- Compound 220 conjugate demonstrated potent tumor growth inhibition. In this tumor model, the isotype controls showed some tumor growth inhibition also, though less than the TBS conjugates did, and the trastuzumab antibody alone had a small growth inhibition effect relative to vehicle control.

FIG. 8 summarizes tumor growth of SKOV3ip xenografts after a single dose of a conjugate of Compound 215 with trastuzumab (TBS-Cmpd 215) at doses of 5 mg/kg and 10 mg/kg. The 5 mg/kg dosage achieved significant growth inhibition, while the 10 mg/kg dose shrank tumors. In this experiment, the 5 mg/kg dose of trastuzumab alone (no Eg5 inhibitor) and the 5 mg/kg dose of isotype control (Compound 215 conjugated to an antibody that does not recognize the SKOV3ip tumor cells) both appear to inhibit tumor growth. Moreover, neither of these was as effective as the TBS-Compound 215 conjugate at comparable dosages.

FIG. 9 summarizes tumor growth of SKOV3ip xenografts after a single dose of a conjugate of Compound 223, which contains Compound 17 as payload, with trastuzumab (TBS-Cmpd 223, or TBS— 5B) at doses of 5 mg/kg and 10 mg/kg. The 5 mg/kg dosage achieved significant growth inhibition, while the 10 mg/kg dose resulted in tumor stasis. In this experiment, the 10 mg/kg dose of trastuzumab alone (no Eg5 inhibitor) modestly inhibited tumor growth. The 5 mg/kg and 10 mg/kg doses of isotype control (Compound 215 conjugated to an antibody that does not recognize the SKOV3ip tumor cells) do not significantly inhibit tumor growth compared to the vehicle treated tumors.

Activity Example 4. Novel Linkers that reduce ADC aggregation.

In this example, an ADC was constructed with a simple linker and a payload compound that produced a significant and undesirable amount of aggregation. The antibody in each of the ADCs used in this example is TBS. The construct referred to as ADC-1 10 uses an unbranched linker and exhibits about 12% aggregation. See Figure 10(A). Modifying the linker of this ADC (see ADC-1 1 1 and ADC-1 12) to introduce a polar group off of the acylated nitrogen reduces aggregation to less than 3%. See Figure 10(B) and Figure 10(C).

The payload and payload plus linker attachment point for each of these ADCs:

ADC-110 ADC-Il l ADC-112

Payload showing attachment point for Linker

Thus ADC-1 1 1 consists of the antibody trastuzumab attached to the maleimide group of Compound 367, and ADC-1 12 consists of the same antibody attached the the maleimide group of Compound 368. ADC-1 10 consists of Compound 366 and trastuzumab. The Linker in these ADCs is considered non-cleavable. These ADCs demonstrate that R¹ in compounds of Formula II, IIC and III can be used as a point of attachment to a target binding moiety, e.g., an antibody even when a non-cleavable linker is used. Figure 1 1 shows in vitro efficacy of ADC-1 10 and ADC-1 1 1 against several cancerous cell lines, demonstrating that the conjugates are highly active against cell lines with high Her2 levels (SKOV3ip and MB231-M16), less active against cells having Her2 but that are less responsive to trastuzumab ADCs due perhaps to Her2 turnover rates (MB231-W6), and much less active against Her2 negative cells (MB468), even though these cells are highly sensitive to Eg5 inhibitors. The SKOV3ip and MDA-MB231-M16 cell lines have high Her2 expression and are thus expected to be sensitive to these trastuzumab conjugates.

MB231-W6 also has Her2 expression, but is less sensitive to trastuzumab ADCs, likely due to the turnover rate for Her2. MDA-MB468 is Her2-negative, but is highly sensitive to Eg5 inhibitors.

Figure 1 1 (A) shows that SKOV3ip cells are highly sensitive to ADC-1 10 and ADC-1 1 1 , which is expected due to the high Her2 level on these cells. The MB468 cell line is much less sensitive, as expected based on its lack of Her2. Figure 1 1 B shows that the MB321- M16 cell line is more sensitive than MB321-W6, which is believed to be due to lower turnover of the Her2 antigen giving less efficient internalization of the ADC in MB321-W6 cells. Figure 1 1 (C) compares SKOV3ip to both of the low sensitivity cell lines, MB468 and MB231-W6. The data in Figure 1 1 also shows that the branched linker in ADC-1 1 1 does not significantly affect its activity relative to the un-branched linker in ADC-1 10. Thus the branched linker reduces aggregation of the ADC without interfering with efficacy.

Similarly, an ADC was made using antibody TBS and Payload/Linker Compound 6D (see Table 6). This ADC has a linker with a carboxylate group directly on the alkyl chain of the linker. An otherwise identical ADC was made that lacked the carboxylate on the linker. The ADC with carboxylic acid group on the linker had DAR = 4.9 and 3% aggregation, while the corresponding ADC lacking the carboxylic acid group on the linker had DAR = 4.2 but aggregation was 1 1.6%. Again, a polar group on the linker reduced aggregation significantly. Both of these ADCs exhibited antibody-dependent inhibition of tumor cell growth in cell culture on Her2+ cell lines, but the ADC with the carboxylic acid on the linker was significantly more active on MB468 cells (breast cancer).

Activity Example 5. Activity of Immunoconjugates targeting other antigens.

Immunoconjugates were prepared from payload compounds shown in Table 5, conjugated with an antibody referred to as cKitA. This antibody recognizes a different antigen from the antibody referred to as trastuzumab— it is selective for the antigen cKit, also referred to as CD1 17. cKit is found on hematopoietic stem cells and progenitor cells, and is associated with mast cell neoplasms, gastrointestinal stromal tumors (GISTs), germ cell tumors and some leukemias. Accordingly, immunoconjugates of the invention having anti-cKit antibodies are useful to treat these conditions.

Conjugates having a drug to antibody ratio (DAR) between 3.0 and 4.5 were prepared with this antibody by the methods described above. The immunoconjugates were tested for activity in a cell line expected to be recognized by the anti-cKit antibody, cKitA. Figure 12 shows inhibition of cell growth by six of these immunoconjugates. While activity varies with payload, all were quite active at concentrations below 1 microgram of immunoconjugate per ml_, with the most active showing potent cell growth inhibition at about 1 ng/mL.

SEQ ID NO:3 (constant region of the heavy chain wild type of antibody

cKitA)

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK

FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

SEQ ID NO:4 (constant region of the light chain wild type of antibody

cKitA)

KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

SEQ ID NO:5 (constant region of the mutant heavy chain of antibody cKitA with mutation HC-S375C)

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK

FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK SEQ ID NO:6 (constant region of the mutant light chain of antibody cKitA with mutation LC-K107C)

CRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN

RGEC

SEQ ID NO: 7 (constant region of the mutant heavy chain of antibody cKitA

with the double mutation HC-E152C-S375C)

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD

KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK

FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI

AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

SEQ ID NO: 8 (constant region of mutant heavy chain antibody cKitA with

mutation at HC360C)

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

REEMTCNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Activity Example 6. Comparison of Immunoconjugates with Linker Variations.

Immunoconjugates were prepared from compounds in Tables 5 and 6 by the methods decribed herein. These compounds contain similar Eg5 inhibitors as payloads, but have different linkers. In each case the payload is linked through the group corresponding to R¹ in Formula II. The antibody referred to herein as TBS was used. The immunoconjugates all had DAR between 3 and 5. These immunoconjugates were tested for cell growth inhibition against four different cell lines expected to vary in sensitivity to the antibody. All cell lines were found to be inhibited by each of the compounds. The variations in the linker within the scope of Formula II as described herein had measurable, but generally modest effects on cell growth inhibition, as illustrated by the inhibition curves in Figure 13. One attachment point for the linker, the nitrogen atom of an azetidine ring at R¹ , did significantly reduce activity under the test conditions. The data demonstrates that a variety of linkers are suitable for use in immunoconjugates of Formula I containing payload compounds of Formula II. Notably, putting a hydroxy group on the phenyl ring corresponding to Ar2 does not significantly increase or decrease activity in cell culture.

Activity Example 7. Comparison of Eg5 Inhibitors of Formula II with other Eg5 Inhibitors as ADC Payloads.

Immunoconjugates were prepared from payloads 6N (payload-linker Cmpd 509) and 6P (Cmpd 508), in Table 2. These payloads are potent inhibitors of Eg5 known in the literature, and represent classes of compounds other than the compounds of Formula II. For comparison, immunoconjugates having a compound of Formula II (Cmpd 220) as payload and containing the same 'val-cit' cleavable linker as 6N and 6P was tested along with the other classes of Eg5 inhibitors. While the other classes of Eg5 inhibitors exhibited activity as payloads, their immunoconjugates were less active by about ten-fold than immunoconjugates having Formula II payload compounds, as illustrated in Figure 14.

In Vivo Activity

General Procedure: Female nu/nu mice (Harlan Laboratories, Livermore, CA) were injected subcutaneously in the right flank with 5x10⁶ SK-OV-3ip1 tumor cells suspended in HBSS in 50% Matrigel™ (BD Biosciences) in a total volume of 200 μΐ. Alternatively, female SCID/Beige mice (Harlan Laboratories, Livermore, CA) were injected subcutaneously in the right flank with 5x10⁶ H526 tumor cells suspended in HBSS in 50% Matrigel™ (BD Biosciences) in a total volume of 200 μί. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8^th edition, 2001 ).

For efficacy studies, mice were randomized between days 7-10 post-implant and animals enrolled in the study with a mean tumor volume of approximately 225 mm³. Typically, at least 5 mice were used for each treatment group— the number of mice per group is indicated in the Figures summarizing test results. Mice were dosed once intravenously with antibody-drug conjugates or vehicle (50 mM citrate, 140 mM NaCI, pH 7.3) via lateral tail vein.

Tumor xenografts were measured in two dimensions (L and W) with digital calipers twice weekly from the start of dosing on Day 0. Tumor volume was calculated as (L x W²)/2.

Body weights were measured twice weekly and clinical observations were recorded daily. Tumor volume and body weights were captured and stored by StudyDirector software (StudyLog, South San Francisco, CA). After 50 days of study duration, animals were humanely euthanized.

Activity Example 8.

An immunoconjugate of formula 5B in Table 5 where, the antibody (AntiB) is trastuzumab was prepared as described herein (TBS-5B). Its activity was compared against an immunoconjugate (TBS-DM1 ) having the same antibody and a known ADC payload (DM1 ), and an immunoconjugate where AntiB is an igG1 kappa chain specific for a viral glycoprotein, gH (immunoconjugate: gH-5B), in cellular assays. Figure 15 shows cell proliferation results for TBS-5B, TBS-DM1 , and gH-5B in a cell line having high Her2 expression (SK-OV-3ip, a human ovarian cancer cell line). The conjugate having 5B as payload was comparable in activity to the one having DM1 as payload, demonstrating the high efficacy of the Eg5 inhibitor as an ADC payload. As expected, the conjugate having gH as the antibody component exhibited very little activity in the same cell line, which demonstrates that the activity of the TBS-5B conjugate is dependent on the TBS antibody. For comparison, 5B-TBS was also tested on a cell line with low Her2 expression. As expected, the immunoconjugate was not active on a cell line lacking the antigen (Her2) targeted by its antibody. This demonstrates that the activity of 5B is dependent on matching the antibody of the immunoconjugate with expression of its targeted antigen on the cells.

The same immunoconjugates were then tested in vivo, on SK-OV-3ip xenograft tumors in mice. This xenograft responds poorly to TBS-DM1 .

Figure 16 shows potent tumor growth inhibition by TBS-5B, an immunoconjugate of the invention. Two control groups were used: one control group was treated with the TBS antibody alone (TBS), and the other was treated with a conjugate of 5B with IgG kappa specific for glycoprotein gH (gH-5B). The TBS-5B immunoconjugate produced a normal dose response and statistically significant tumor inhibition relative to the vehicle and gH controls by day 20 using the indicated standard. At the 10 mg/kg dose, almost no tumor growth occurred out to 30 days. Each group had 9 mice, and none of the groups showed significant body weight loss during treatment. Tumor growth inhibition by the controls was low, and was not statistically significant under the conditions of the test, using the indicated standard. These results show that the Eg5 inhibitor is an effective ADC payload for use in vivo to treat a tumor targeted by its antibody, and that efficiacy depends on both the Eg5 inhibitor and an antibody matched with the cell line.

Figure 17 illustrates that the Eg5 immmunoconjugates are more active in vivo on the SK-OV-3ip xenograft tumors in mice than a conjugate containing a payload used in ADCs in clinical trials (DM1 ) attached to the same antibody (TBS). Again, tumor growth was almost completely stopped by the TBS-5B immunoconjugate at 10 mg/kg dose and was statistically significant relative to control by 30 days, using the indicated standard. A comparable dose of TBS-SMCC-DM1 was less effective, not reaching statistical significance by 30 days. Treatments with antibody alone (TBS) or with either DM1 or 5B attached to the gH viral glycoprotein IgG were much less active. This demonstrates that the Eg5 inhibitor payload is at least as effective as other payload classes that have been successful in clinical trials (DM1 is the payload for the FDA-approved immunoconjugate Kadcyla®), and that its in vivo efficacy is dependent on specific recognition of the targeted tumor cells by the antibody of the immunoconjugate.

Activity Example 9: In Vivo Efficacy with Other Antibodies

Immunoconjugates were prepared containing the Eg5 inhibitors of the invention linked to antibodies specific for the cKit antigen, referred to herein as 'cKit antibodies'. The cKit conjugates were tested on human small-cell lung cancer xenograft tumors (H526) in mice. (n=5; no significant body weight loss for any group) Figure 18 shows H526 tumor growth inhibition by an immunoconjugate having payload-linker 5B on a first cKit antibody (cKitA) at a dose of 6.5 mg/kg. Lower doses were much less active, and neither the cKitA antibody alone nor the Eg5 payload-linker combination (5B) conjugated to an antigen-binding group specific for the viral glycoprotein gH exhibited measurable tumor growth.

Figure 19 summarizes results of a comparison of the cKitA immunoconjugate with an Eg5 inhibitor (5B) and SMCC-DM1 (n=5 for each curve; no significant body weight loss in any group). Inhibition of tumor growth in H526 xenograft tumors in mice is shown for cKitA immunoconjugates with either 5B or SMCC-DM1 attached. The SMCC-DM1 conjugate exhibited statistically significant inhibition of tumor growth at the 10 mg/kg dose, but not at 5 mg/kg, using the indicated standard. The 5B conjugate gave inhibition at 5 mg/kg that was statistically significant and comparable to the 10 mg/kg dose of the SMCC-DM1 conjugate, and the 5B (Eg5 inhibitor) conjugate was much more active at the 10 mg/kg dose. Controls showed no activity for a conjugate of 5B with gH, which is specific for a viral glycoprotein gH. Activity Example 10. Cross-Over Experiment.

For this experiment, each mouse was implanted with two different xenograft tumors, SK-OV-3ip on one side of the body, and H526 on the other side. The SK-OV-3ip cell line is Her2-positive (Her2+) and lacks cKit (cKit-), while the H526 cell line is Her2- negative (Her2-) and cKit positive (cKit+). Each mouse was then treated with vehicle or one of three immunoconjugates: gH-5B, TBS-5B or cKitA-5B. As Figure 20 shows, the [Her2+, cKit-] tumor growth was inhibited strongly by TBS-5B, as expected due to its expected binding to the TBS antibody. None of the other treatments significantly affected tumor growth. Similarly, the [Her2-, cKit+] tumor growth was inhibited only by the cKit-5B conjugate and was not affected by any of the other treatments. This cross-over experiment proves that tumor inhibition is due to the immunoconjugate, and does not occur from either antibody or released payload— only the intact immunoconjugate whose antibody matches the antigen on the tumor is effective.

Activity Example 11. Impact of Linker Variations

This example illustrates the effect of linker variations on in vivo efficacy of immunoconjugates having similar Eg5 inhibitors as payloads (5B, 5H, 5G, and 5A— see Table 5). Four immunoconjugates were prepared using different linkers to attach similar Eg5 inhibitors to trastuzumab antibodies. Figure 21 summarizes the activity of the immunoconjugates, which all were similarly effective at inhibition of SK-OV-3ip tumor xenografts in mice at a dose of 10 mg/kg. These immunoconjugates were also similar in activity to an immunoconjugate of the same antibody (TBS) with MMAF, a payload used in ADCs in clinical trials (TBS-MC-MMAF), and were more active than an

immunoconjugate with a maytansine payload on the same antibody (TBS-SMCC-DM 1 ). As expected, controls using the same payloads conjugated to an antibody specific for a viral glycoprotein (gH), and unconjugated antibody TBS, exhibited little or no tumor growth inhibition. (9 mice per group (n=9) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 12. Activity of Diverse Eg5 Payload/Linker Combinations in vivo

This example compares different linker attachment points and linkers, comparing the in vivo efficacy of immunoconjugates having Eg5 inhibitors as payloads with linkers attached at two different positions, using two different linkers for each attachment point (5B, 5E, 5F, and 5D— see Table 5), all conjugated with trastuzumab antibodies. Figure 22 summarizes the activity of the immunoconjugates, which all inhibited growth of SK-OV- 3ip tumor xenografts in mice at a dose of 10 mg/kg. These immunoconjugates were similar in activity, and were equal or better when compared to a TBS-SMCC-DM1 conjugate. As expected, controls using the unconjugated antibody (TBS) exhibited little growth inhibition. (8 mice per group (n=8) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 13. Activity of Diverse Eg5 Immunoconjugates on NCI-N87 Xenografts

This example compares in vivo efficacy of immunoconjugates having various Eg5 inhibitors as payloads with linkers on a tumor cell line (NCI-N87, a gastric tumor line) that was more difficult to inhibit. Four immunoconjugates with Eg5 inhibitor payloads (5B, 5E, 5D, and 6U— see Tables 5-6) were prepared using different Eg5 inhibitor-payload combinations, all conjugated with trastuzumab antibodies. Figure 23 summarizes the activity of the immunoconjugates, which all inhibited growth of N87 tumor xenografts in mice at a dose of 10 mg/kg. These immunoconjugates varied in activity in this model, and are compared to a Trastuzumab-SMCC-DM1 immunoconjugate. All of the immunoconjugates appear to inhibit tumor growth; 5B and 6U and the DM1 conjugate achieved statistical significance under the test conditions, using the indicated standard. As expected, controls using the unconjugated antibody (TBS) exhibited little growth inhibition. (8 mice per group (n=8) for each immunoconjugate tested; no significant weight loss in any group).

Activity Example 14. Activity of Eg5 Immunoconjugates on H526 Xenografts

This example compares in vivo efficacy of immunoconjugates having various Eg5 inhibitors as payloads conjugated with a cKit antibody on a tumor cell line that expresses cKit (H526). Six immunoconjugates with Eg5 inhibitor payloads (5B, 5E, 5F, 5C, 5A, and 5D— see Table 5) were prepared using different Eg5 inhibitor-payload combinations, all conjugated with a cKit antibody (cKitA). Figure 24 summarizes the activity of the immunoconjugates, which all inhibited growth of H526 tumor xenografts in mice at a dose of 5 mg/kg. (5 mice per group (n=5) for each immunoconjugate tested; no significant weight loss in any group). Payload/linker combinations 5E and 5D were more potent at the 5 mg/kg dose, achieving statistically significant tumor growth inhibition using the indicated standard. Figure 25 summarizes the activity of the same immunoconjugates on H526 xenografts at 10 mg/kg. (5 mice per group (n=5) for each immunoconjugate tested; no significant weight loss in any group). The immunoconjugates varied in activity at 10 mg/kg in this model, with 5D appearing to be most active and long lasting; both 5D and 5E achieved statistically significant tumor growth inhibition at this dose using the indicated standard.

Activity Example 15. Comparison of cKit Antibodies in Eg5 Inhibitor Conjugates

This example compares in vivo efficacy of immunoconjugates having different cKit antibodies with an Eg5 inhibitor payload, on a tumor cell line that expresses cKit (H526). Beginnning with cKit A, two modified cKit antibodies were prepared by the following general method. Cys mutants of the cKitA antibody were expressed in 293 Freestyle™ cells by co-transfecting heavy chain and light chain plasmids using transient transfection method as described previously (Meissner, et al., Biotechnol Bioeng. 75:197-203 (2001 )). The DNA plasmids used in co-transfection were prepared using Qiagen plasmid preparation kit according to manufacturer's protocol. 293 Freestyle™ cells were cultured in suspension in Freestyle™ expression media (Invitrogen) at 37°C under 5% C0₂. On the day before transfection, cells were split to 0.7 x 10⁶ cells /ml into fresh media. On the day of transfection, the cell density typically reached 1.5 x 10⁶ cells/ml. The cells were transfected with a mixture of heavy chain and light chain plasmids at the ratio of 1 : 1 using PEI method (Meissner et al., 2001 ). The transfected cells were further cultured for five days. The media from the culture was harvested by centrifugation of the culture at 2000x g for 20 min and filtered through 0.2 micrometer filters. The expressed antibodies were purified from the filtered media using Protein A-Sepharose™ (GE Healthcare Life Sciences). Antibody IgGs were eluted from the Protein A-Sepharose™ column by the elution buffer (pH 3.0) and immediately neutralized with 1 M Tris-HCI (pH 8.0) followed by a buffer exchange to PBS.

Engineered Cys ADCs have been reported to be better tolerated in mice and rat animal models than ADCs made by conjugation to partially reduced native disulfides or through native lysine residues. To evaluate differences in in vivo efficacy between ADCs conjugated through engineered Cys antibodies and ADCs conjugated to partially reduced native disulfide bonds, an Eg5 linker-payload Compound 223 was conjugated to antibody cKitA HC-E152C-S375C double mutant (cKitB: the immunoconjugates are referred to as cKitB— Cmpd 223 or cKitB— 5B) and cKitA HC-K360C-LC-K107C double mutant (cKitC: immunoconjugates are referred to as cKitC— Cmpd 223 or cKitC— 5B) as well as wild- type cKitA antibody (immunoconjugates cKitA— Cmpd 223 or cKitA— 5B). (Residue Numbers are EU numbers)

The engineered antibodies contain newly-introduced cysteine residues at specific locations found to be particularly suitable for payload attachment. Antibody cKitB is a modified version of cKitA having two cysteine residues engineered into its heavy chain, and cKitC is a modified version of cKitA having one cysteine residue engineered into its heavy chain and one into its light chain. Because cKitB and cKitC each have two heavy chains and two light chains, the modified antibodies have 4 newly-added cysteine residues useful for conjugation without need for reduction of the interchain disulfides, so immunoconjugates having four payload groups attached (DAR = 4) can be prepared. Each cKit antibody was conjugated with payload/linker combination 5B using the methods described herein in order to compare the effect of modifying the antibody sequence.

Antibody cKitA and mutants cKit B and cKitC were prepared following protocols described in Example 5. cKitB and cKitC were reduced and reoxidized following the protocol describe in Example 6. Reoxidized antibodies were conjugated with Compound 223 by incubating 5 mg/ml antibody with 0.35 mM compound 223 for 1 hour in 50 mM sodium phosphate buffer (pH 7.2). The completeness of the reaction was monitored by RP-HPLC and a DAR of 3.9 and 4.0 were obtained for the cKitB and cKitC conjugates, respectively. DAR measurements were further verified by MS. ADCs were shown to be potent and in vitro cell killing assays and had pharmacokinetics properties similar to unconjugated antibody in non-tumor bearing mice.

The ADC with Compound 223 conjugated to the native disulfide bonds of cKitA was prepared as follows in a 2-step process. The antibody at a concentration of 5-10 mg/ml in PBS containing 2 mM EDTA, was first partially reduced for 1 hour at 37 °C with 50 mM mercaptoethylamine (added as a solid). After desalting and addition of 1 % w/v PS-20 detergent, the partially reduced antibody (1 -2 mg/ml) was reacted overnight at 4 °C with an amount of 0.5-1 mg of Cmpd223, dissolved at 10 mg/ml in DMSO, per 10 mg antibody. The ADC was purified by Protein A chromatography. After base-line washing with PBS, the conjugate was eluted with 50 mM citrate, pH 2.7, 140 mM NaCI, neutralized and sterile filtered. The average DAR was 3.2.

Properties of the three cKit ADCs: cKitA— 5B: DAR = 3.2, aggregation 0.8% cKitB— 5B: DAR = 3.9, aggregation 1.5% cKitC— 5B: DAR = 4.0, aggregation 3.2%

Immunoconjugates with the following combinations of payloads with cKit and trastuzumab antibodies and mutated antibodies were prepared and characterized by the same methods. Note that the engineered antibodies consistently provided DAR near 4, the expected loading if the four added cysteine residues per antibody complex are all conjugated to payload:

Figure 26 summarizes the activity of two of the immunoconjugates made with cysteine-engineered cKit antibodies, which inhibited growth of H526 tumor xenografts in mice at doses of 5 mg/kg and 10 mg/kg. (6 mice per group (n=6) for each

immunoconjugate tested; no significant weight loss in any group). Possibly because the engineered antibodies can form conjugates without disturbing the native disulfide bridged structure, their immunoconjugates were more active than the conjugate of cKitA at both doses. Thus, while immunoconjugates of Eg5 inhibitors were active with various cKit antibodies including unmodified ones, this demonstrates that protein engineering to introduce new cysteine residues into the constant regian and using the new cysteine residues as attachment points for the payload/linker group can provide improved immunoconjugates.

Additional Activity Data for Tumor Cell Growth Inhibition

Figure 28 provides additional data demonstrating that immunoconjugates of the invention inhibit growth of tumor cells. Data in Figure 28 demonstrates that these

immunoconjugates inhibit growth of a variety of tumor cell lines, including SK-OV-3ip, MDA-MB-231 , HCC1954, MDA-MB-468, MDA-MB-231 -M40, MDA-MB-231 -M16,H526, and NCI-N87. In each case, the activity of the ADC is partly determined by the

expression level of the antigen recognized by the antibody of the ADC on the treated cells, as expected. The additional data demonstrates that structural variations representative of the scope of Formula (II) generally produce active ADCs, even though the level of activity varies across cell lines. The data further demonstrates that a wide variety of linkers can be used, and that linkers can be attached at positions on groups R1 , Y and Q.

A summary of the in vitro cell growth inhibition by various ADCs of the invention is provided in the following Table. The first column lists the compound ID number from Table 2, 5 or 6 for the payload/linker combination used in the ADC; the second column indicates which antibody is used in the ADC. The third column identifies the cell line on which the ADC is being tested: for most of the ADCs, activity was determined against both an antigen-high cell line, where robust activity is expected, and against an antigen- low cell line where antibody delivery is expected to be inefficient, so activity is expected to be much lower. Activity is reported as Absolute AC50 (ng/mL), Ainf (%), and Relative EC50 (ng/mL). Information about the method used for this data is provided after the table.

Antigen- high Antigen-low

Abs

Abs Rel AC5 Rel AC50 EC50 0 EC50

Cmp Antib (nq/m Ainf (nq/ (nq/ Ainf (nq/mL d ID odv Cell line U mU Cell line mU 1

100. MDA- 3770

Calu-3 550.0 57.5 8 MB-486 .4 75.4 4659.7

6902

HCC1954 20.6 84.2 17.7 PC-3 .9 60.5 6917.7

NCI- H2170 10.9 92.4 10.3

MDA-MB- 231 -M16 37.8 53.9 14.9

NCI-N87 60.6 77.2 43.9

UACC- 812 27.0 64.5 20.3

SK-OV- NCI- >900

5B TBS 3ip 28.6 67.4 14.1 H526 0 20.3 >9000

MDA- >900

HCC1954 21 .9 88.1 20.7 MB-231 0 -0.9 >9000

MDA-MB- MDA- 3638 231 -M16 550.9 55.7 56.1 MB-486 .2 83.4 3423.0

OE19 78.0 72.3 57.9

1 19.

NCI-N87 615.0 64.8 1

1834. >900 MDA- >900

5B cKitA NCI-H526 5 76.5 0 MB-468 0 0.5 >9000

SK-OV- MDA- >900

5A TBS 3ip 196.8 58.4 25.2 MB-231 0 2.1 >9000

MDA- 8171

HCC1954 30.6 79.1 24.9 MB-486 .2 13.6 8171 .2

MDA-MB- 231 -M16 201 .6 55.1 63.0

MDA- >900

5A cKitA NCI-H526 40.6 85.0 29.8 MB-486 0 16.5 >9000

SK-OV- >900 1499 MDA- >900

6G TBS 3ip 0 38.2 .9 MB-231 0 -8.3 >9000 Antigen- high Antigen-low

Abs

Abs Rel AC5 Rel

AC50 EC50 0 EC50

1014. 351 . MDA- 8656

HCC1954 7 62.1 0 MB-486 .9 36.4 >9000

MDA-

MB-

MDA-MB- 231 - >900

231 -M16 37.4 58.6 17.4 M40 0 -9.9 >9000

MDA- >900

5C TBS HCC1954 343.8 70.4 65.1 MB-231 0 -1 .6 >9000

SK-OV- >900 MDA- 7340

3ip 0 41 .3 43.7 MB-486 .0 50.9 5672.2

MDA-MB-

231 -M16 226.3 57.9 56.6

4527 MDA- >900

5C cKitA NCI-H526 268.6 78.3 .8 MB-486 0 35.5 8272.5

SK-OV- >900 >900 MDA- 5853

452 TBS 3ip 0 26.3 0 MB-468 .1 56.4 6259.6

7173. >900

HCC1954 7 53.7 0

SK-OV- MDA- 2033

458 TBS 3ip 632.5 66.1 76.9 MB-468 .6 78.1 1726.7

HCC1954 108.8 84.3 61.4

SK-OV- 181 . MDA- 179.

460 TBS 3ip 328.9 71 .8 9 MB-468 3 88.0 179.0

SK-OV- 136. MDA- 159.

461 TBS 3ip 290.4 73.5 7 MB-468 2 88.0 148.1

SK-OV- 261 . MDA- 143.

463 TBS 3ip 409.3 70.5 5 MB-468 8 87.1 136.0

SK-OV- 365. MDA- 171 .

462 TBS 3ip 476.8 73.6 4 MB-468 0 88.4 170.1

SK-OV- 1766. >900 MDA- 5233

446 TBS 3ip 8 57.2 0 MB-468 .0 75.0 5209.6 Antigen- high Antigen-low

Abs

Abs Rel AC5 Rel

AC50 EC50 0 EC50

129.

TBS HCC1954 347.2 65.6 3

SK-OV- MDA- >900

5D TBS 3ip 193.2 51 .0 19.3 MB-468 0 10.2 >9000

MDA-

MB-

231 - >900

HCC1954 79.5 81 .4 29.5 M40 0 -22.1 >9000

MDA- 821 1

5D cKitA NCI-H526 29.7 86.5 20.8 MB-468 .5 55.3 821 1 .5

MDA-

MB-

SK-OV- 231 - >900

465 TBS 3ip 90.3 72.9 17.7 M40 0 -5.5 >9000

MDA- 1031

HCC1954 1 15.1 82.2 55.4 MB-468 .4 77.1 877.4

MDA- 8067

6A TBS HCC1954 31.2 82.6 20.3 MB-231 .6 59.0 8067.6

MDA-

MB-

SK-OV- 231 - >900

3ip 82.3 67.0 10.5 M40 0 38.1 >9000

MDA-MB- MDA- 1293

231 -M16 149.9 56.6 48.7 MB-468 .6 81 .7 1 164.9

MDA-

MB-

SK-OV- 1480. >900 231 - >900

384 TBS 3ip 9 63.5 0 M40 0 -14.4 6236.2

MDA- 5329

HCC1954 142.8 69.0 52.6 MB-468 .4 57.0 5736.8

SK-OV- >900 >900 MDA- >900

385 TBS 3ip 0 17.6 0 MB-468 0 10.0 >9000

>900 >900

HCC1954 0 34.9 0

Antigen- high Antigen-low

Abs

Abs Rel AC5 Rel AC50 EC50 0 EC50

SK-OV- MDA- 2217

485 TBS 3ip 22.9 66.9 9.7 MB-468 .9 87.3 2032.9

SK-OV- >900 >900 MDA- >900

393 TBS 3ip 0 -3.3 0 MB-468 0 -6.2 >9000

SK-OV- MDA-

489 TBS 3ip 18.2 74.3 12.4 MB-468 48.5 89.7 46.0

SK-OV- MDA- 902.

486 TBS 3ip 46.8 70.1 24.9 MB-468 6 87.7 833.7

SK-OV- >900 >900 MDA- >900

371 TBS 3ip 0 3.3 0 MB-231 0 7.9 >9000

>900

NCI-N87 130.8 37.6 0

SK-OV- MDA- >900

487 TBS 3ip 123.3 61 .4 13.9 MB-231 0 3.1 >9000

NCI-N87 237.5 62.4 35.3

SK-OV- MDA- 8520

451 TBS 3ip 10.0 84.8 6.0 MB-231 .0 51 .8 6060.0

NCI-N87 231.9 66.2 44.6

MDA- >900

5B cKitB NCI-H526 19.9 81 .3 17.5 -3.9 >9000

MB-468 0

MDA- >900

5B cKitC NCI-H526 27.6 79.0 21 .6 -1 .9 >9000

MB-468 0

Cell Lines used in the preceding table: cell line lineaae

Calu-3 lung

CMK-1 1-5 hematopoietic

HCC1954 breast

HCC70 breast

HT-29 colon

MDA-MB-231 Breast

MDA-MB-231-M16 Breast

MDA-MB-231 -M40 Breast

MDA-MB-486 breast

NCI-H2170 lung

NCI-H526 lung

NCI-N87 gastric

OE19 ovary

PC-3 prostate

SK-OV-3ip ovary

UACC-812 breast

Definition and derivation of potency values:

Cell TiterGlo 2 data were averaged for replicate samples then normalized to untreated cells. Assays included wells of untreated cells, reflecting uninhibited cell growth. The means of the controls were used to normalize the results from treated samples to a % scale. Analysis of the normalized data was done using a 4 parameter logistical model with a standard assay data analysis software (Helios software application).

The 4 parameters include the Ainf (plateau of maximal activity, generally found at high [ADC]), the AO (plateau of minimal activity, generally found at low [ADC]), the concentration of ADC at the midpoint between these two plateaus on the y-axis, and n the slope of the fitted curve at the midpoint between the two plateaus.

For each compound the software derived 3 metrics, the Absolute AC50, the Relative EC50, and the Ainf. The Absolute AC50 ("Abs AC50" in the table) is the concentration of ADC where the fitted curve crosses 50% on the Y-Axis. The Relative EC50 ("Rel EC50" in the table) is the concentration of ADC at the midpoint of the fitted curve between AO and Ainf.

Comparative Immunoconiuqates:

TBS-SMCC-DM1 : antibody = trastuzumab; linker = SMCC; payload = DM1 : DAR is about 3.5

MC-MMAF Conjugate, which uses a maleimidocaproyi linker:

Claims

1 . An immunoconjugate of Formula (I):

(I)

wherein Ab represents an antigen binding moiety;

L represents a linking group that connects X to Ab;

m is an integer from 1 -4;

n is an integer from 1 to 16; and

X independently at each occurrence represents a group of Formula (II)

(II)

that is connected by L to Ab, wherein:

Z is N or CH;

Ar¹ is phenyl optionally substituted with up to three groups selected from halo, Ci_₃ alkyl, and C1-3 haloalkyl;

Ar² is phenyl or pyridinyl, and Ar² is optionally substituted with up to two groups selected from halo, CN, C1.3 alkyl, hydroxyl, amino, and C1.3 haloalkyl;

R¹ is C1-6 alkyl, -(CH₂)₀-2-C₃-6 cycloalkyl, or -(CH₂)o-2-C₄-7 heterocyclyl containing up to two heteroatoms selected from N, O and S as ring members, wherein each Ci_-6 alkyl, C3-6 cycloalkyl, or C₄-₇ heterocyclyl is optionally substituted with up to three groups selected from halo, C1-4 alkyl, C1-4 haloalkyl, C1-4 alkoxy, hydroxyl, amino, carboxy, oxo, hydroxyl-substituted Ci_₄ alkyl, amino-substituted C_1-4 alkyl, -C(0)-Ci_₆ alkyl, -C(0)-NH-Ci_₆ alkyl -C^O-d-e alkyl, and COO(d-₄ alkyl);

R² is H or d_ 4 alkyl;

T is (CH₂)₁_₃;

Y is selected from Ci_₃ aminoalkyl, C₄.₆ heterocyclyl, and C₃.₆ cycloalkyl, wherein C₁-3 aminoalkyl, C₄.₆ heterocyclyl, and d-₆ cycloalkyl are each optionally substituted with up to three groups selected from amino, oxo, halo, hydroxyl, Ci_₄ alkyl, Ci_₄ alkoxy, hydroxyl-substituted Ci_₄ alkyl, amino-substituted Ci_₄ alkyl, COOH, COO-(Ci_₄ alkyl), -

alkyl)₂, and Ci_₄ haloalkyl;

A is NH, N(Ci-₄ alkyl), or a bond between the carbonyl in Formula (II) and Q;

Q is selected from d-₄ alkyl, -0-d-₄alkyl, -(CH₂)o-₂-C₄.₆heterocyclyl, -(CH₂)₀.₂-d- ecycloalkyl, -(CH₂)o-₂-C₅-6heteroaryl, and -(CH₂)₀.₂-phenyl, and is optionally substituted with up to three groups selected from halo, hydroxyl, amino, -SH, -R, -OR, -SR, -S0₂R, -NHR, -O-glucuronate, and -NR₂, where each R is Ci_₆ alkyl, C₃.₆ cycloalkyl, or a 4-6 membered heterocycle containing N, O or S as a ring member, and each R is independently optionally substituted with halo, -SH, -NH₂, OMe, or -OH.

2. The immunoconjugate according to claim 1 , wherein R² is H.

3. The immunoconjugate according to claim 1 or claim 2, wherein Z is CH.

4. The immunoconjugate according to claim 1 or claim 2, wherein Z is N.

5. The immunoconjugate according to any one of claims 1 to 4, wherein R¹ is tetrahydropyran, and R¹ is optionally substituted with up to two groups selected from oxo and methyl.

6. The immunoconjugate according to any of the preceding claims, wherein Ar¹ is dihalophenyl.

7. The immunoconjugate according to any of the preceding claims, wherein the compound of Formula (II) has the formula:

wherein L is attached to Y, or to Q, or to R¹.

8. The immunoconjugate according to any of the preceding claims, wherein R¹ is 4- tetrahydropyranyl.

9. The immunoconjugate according to any of claims 1 -8, wherein R¹ is -C(Me)₂- (CH₂)o-2R³⁰, wherein R³⁰ is -OH, COOH, or NH₂, and L is attached to R¹.

10. The immunoconjugate according to any of the preceding claims, wherein Q is Ci_₄ alkyl substituted with one or two groups selected from hydroxyl and amino.

1 1 . The immunoconjugate according to any of the preceding claims, wherein Y is pyrrolidone optionally substituted with halo, amino, or hydroxy.

12. The immunoconjugate according to any of the preceding claims, wherein A is - NH-.

13. The immunoconjugate of any of the preceding claims, wherein the linking group is cleavable.

14. The immunoconjugate of any of claims 1 -12, wherein the linking group is non- cleavable.

15. A compound of Formula (III):

Ar¹

Y

Q

(I I I)

or a pharmaceutically acceptable salt thereof, wherein: Z is N or CH;

R¹ is -(CH₂)o-2-C₄-7 heterocyclyl or -(CH₂)o-2-C₃.₇ cycloalkyl, where the C₄-₇ heterocyclyl contains up to two heteroatoms selected from N, O and S as ring members, and C4-7 heterocyclyl and C₃.₇ cycloalkyl are each optionally substituted with up to three groups selected from halo, d-4 alkyl, d-4 alkoxy, hydroxyl, amino, oxo, hydroxyl- substituted C1-4 alkyl, amino-substituted C1-4 alkyl, C1-4 haloalkyl, and COO(d-4 alkyl); or R¹ is C₃_6 alkyl substituted with -OH, -COOH or -NH₂;

R² is H or d_ 4 alkyl;

T is (CH₂)₁.₃;

Y is selected from d-2 aminoalkyl, C₄.₆ heterocyclyl, and C₃.₆ cycloalkyl, wherein C1-2 aminoalkyl, C₄-e heterocyclyl, and C₃.₆ cycloalkyl are each optionally substituted with up to three groups selected from amino, oxo, halo, hydroxyl, C1-4 alkoxy, hydroxyl- substituted d-4 alkyl, amino-substituted d-4 alkyl, COOH, COO-(d-4 alkyl), CONH(d_₄ alkyl), CON(d_₄ alkyl)₂, and d_₃ haloalkyl;

A is NH, N(d_ alkyl), or a bond between the carbonyl in Formula (I I I) and Q;

Q is selected from d-4 alkyl, -(CH₂)₀.₂-C₄-₆heterocyclyl, -(CH₂)₀-₂-C₅-₆heteroaryl, and -(CH₂)₀-₂-phenyl, and Q is optionally substituted with up to three groups selected from halo, hydroxyl, amino, -SH, -R, -OR, -SR, -S0₂R, -NHR, -N₃, and -NR₂, where each R is C1-6 alkyl optionally substituted with up to three groups selected from halo, -SH, -NH₂, OMe, and -OH.

16. The compound of claim 15, wherein R¹ is tetrahydropyranyl.

17. The compound of claim 15, wherein R¹ is a group of the formula -C(Me)₂-(CH₂)₀. ₂R³⁰, wherein R³⁰ is -OH, COOH, or NH₂ .

18. A compound of Formula (MA) or (MB) or (IIC):

Or (IIC) wherein Ar¹, Ar², Z, R¹ , R², T, Q, Y, and A are as defined in claim 1 ,

Q^* is selected from -CH₂-, -CH(Me)-, -CH(Me)CH₂-, -CH₂CH₂-, -CH₂0-, -CH₂S-, - CH₂-NH-, -CH₂-NMe-, -CH(Me)0-, -CH(OH)-CH₂0-, -CH(CH₂OH)-0-, -CH(OH)-CH₂NH-, - CH(CH₂OH)-NH-, -CH(CH₂NH₂)-0-, -CH(CH₂OH)-NH-, -CH(Me)S-, -CH(Me)NH- -CH₂CH₂O-, -CH₂CH₂NH-, -CH₂CH₂S-, -CH(Me)CH₂0-, -CH(Me)CH₂S- -CH(Me)CH₂NH-,

, and

Y* is selected from -CH(CH₂F)NH-, -CH₂NH-,

, and where R¹⁰ and R¹¹ are independently H, Me, OMe, F, CH₂F, CH₂OH, COOH, CONH(d_₄ alkyl), CON(Ci-₄ alkyl)₂, COO(Ci-₄ alkyl), or OH,

R^r is selected from C₃.₆ alkyl substituted with hydroxy, amino or carboxy; and W is a linking moiety that comprises one or more linker components and a reactive functional group.

19. The compound of claim 16, wherein W comprises a reactive functional group selected from -SH, -NH₂, -C(=0)H, -C(=0)Me, N-maleimide, -NHC(=0)-CH₂-halo, - COOH, and -C(=0)-OR', wherein halo is selected from CI, Br and I, and -OR' is the leaving group moiety of an activated ester.

20. The compound of any of claims 15-19, wherein Ar¹ is dihalophenyl.

21. The compound of any of claims 15-20, wherein Ar² is phenyl or halophenyl or hydroxyphenyl.

22. The compound of any of claims 15-21 , wherein Z is CH.

23. The compound of any of claims 15-21 , wherein Z is N.

24. The compound of any of claims 15 or 18-23, wherein R¹ is 4-tetrahydropyranyl.

25. The compound of any of claims 15-24, wherein R² is H.

26. The compound of any of claims 15-25, wherein A is -NH-.

27. The compound of any of claims 15-25, wherein A is a bond.

28. The compound of any of claims 15-27, wherein T is CH₂ or CH₂CH₂.

29. The compound of any of claims 15-28, wherein Y is selected from -CH(CH₂F)NH₂,

, and where R¹⁰ and R¹¹ are independently H, Me, OMe, F, CH₂F, CH₂OH, COOH, COO(d_₄ alkyl), or OH.

30. The compound of any of claims 15-29, wherein Q is selected from -CH₂OH, -CH₂-NH₂, -CH(Me)OH, -CH(OH)-CH₂OH, -CH(OH)-CH₂NH₂, -CH(NH₂)-CH₂OH, - CH(NH₂)-CH₂OH, -CH(Me)SH, -CH(Me)NH₂, -CH₂CH₂OH, -CH₂CH₂NH₂, -CH₂CH₂SH, -

CH(Me)CH₂OH, -CH(Me)CH₂SH, -CH(Me)CH₂NH₂, , and

31 . The compound of claim 15, which is selected from the compounds in Table 1 and the pharmaceutically acceptable salts thereof.

32. A pharmaceutical composition comprising a compound of any of claims 15-31 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

33. A combination comprising a therapeutically effective amount of a compound according to one of claims 15-31 or a pharmaceutically acceptable salt thereof and one or more therapeutically active co-agents.

34. A method of treating a cell proliferation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an immunoconjugate of any of claims 1 -14, or a compound of any of claims 15-17, or a pharmaceutically acceptable salt thereof.

35. A compound according to any one of claims 15-17 or an immunoconjugate of any of claims 1 -14 or a pharmaceutically acceptable salt thereof, for use as a medicament.

36. The compound according to claim 35 or a pharmaceutically acceptable salt thereof, wherein the medicament is for use in the treatment of cancer.

37. An immunoconjugate of any of claims 1 -14 or a pharmaceutically acceptable salt thereof, for use to treat cancer.

38. An immunoconjugate Ab-L*-X, comprising a payload (X) linked to an antibody (Ab), wherein the linking group L* comprises a group of the formula -C(0)NR²¹- or -NR²¹-C(0)- wherein R²¹ is of the formula -(CH₂)i-4-R²², where R²² is a polar group selected from -OH, -NH₂, N(R²³)₂, COOR²³, CON(R²³)₂, -(OCH₂CH₂0)_k-OCH₂CH₂OR²³, and -S0₂R²³, where k is 0 to 4 and each R²³ is independently H or Ci_₄ alkyl.

39. An immunoconjugate of Formula (I):

(I)

wherein Ab represents an antigen binding moiety; each L represents an independently selected linking group that connects X to Ab;

m is an integer from 1 -4;

n is an integer from 1 to 16; and

X independently at each occurrence represents an inhibitor of Eg5.

40. The immunoconjugate of claim 39, wherein X is a compound selected from Table 1 .

41 . The immunoconjugate of claim 39, wherein m is 1 and the immunoconjugate is formed by reaction of Ab with a compound selected from Table 2.

42. A compound selected from Table 5 or an immunoconjugate thereof.

43. An immunoconjugate selected from the immunoconjugates in Table 5, wherein AntiB represents an antibody.

44. An immunoconjugate of any one of claims 1 -14 or 38-43, wherein the antigen binding moiety is an antibody having at least one non-native cysteine residue introduced into the constant region, where the linking group L is attached to the non-native cysteine residue.

45. The immunoconjugate of claim 44, wherein m is 1 and n is between 1 and 5, preferably about 2 or about 4.